JP2020518233A - Anti-PD-L1 antibody and use thereof - Google Patents

Abstract

An anti-PD-L1 antibody or antibody fragment is provided. The antibody or antibody fragment specifically binds to the immunoglobulin C domain of the PD-L1 protein. In various examples, the antibody or antibody fragment is VH CDR1 of SEQ ID NO:1, VH CDR2 of SEQ ID NO:116, VH CDR3 of SEQ ID NO:117, VL CDR1 of SEQ ID NO:4, VL CDR2 of SEQ ID NO:5. , And VL CDR3 of SEQ ID NO: 6, or a variant of each thereof. Also provided are methods of using the antibodies or antibody fragments for treating and diagnosing diseases such as cancer and infectious diseases.

Description

Programmed cell death ligand 1 (PD-L1), also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1), is associated with other factors such as pregnancy, tissue allografts, autoimmune diseases, and liver inflammation. Is a 40 kDa type 1 transmembrane protein that is believed to play a major role in the suppression of the immune system during certain events, such as the pathology of Escherichia coli. Binding of PD-L1 to PD-1 or B7.1 transduces an inhibitory signal that reduces proliferation of CD8+ T cells in lymph nodes. In addition, PD-1 can also regulate the accumulation of foreign antigen-specific T cells in lymph nodes through apoptosis further mediated by downregulation of the Bcl-2 gene.

Upregulation of PD-L1 has been shown to allow cancer to evade the host's immune system. Analysis of tumor specimens from patients with renal cell carcinoma revealed that high tumor expression of PD-L1 was associated with increased tumor progression and increased risk of death. Many PD-L1 inhibitors are under development as cancer immunotherapy and they have shown good results in clinical trials.

Inhibition of PD-L1 has the potential to treat infections in addition to treating cancer. In a mouse model of intracellular infection, Listeria monocytogenes induced PD-L1 protein expression in T cells, NK cells, macrophages. Blocking PD-L1 (eg, using blocking antibodies) resulted in increased mortality in infected mice. The block decreased the production of nitric oxide and TNFα by macrophages, decreased the production of granzyme B by NK cells, and decreased the proliferation of L monocytogenes antigen-specific CD8 T cells (provided that the proliferation of CD4 T cells was increased). Did not decrease). This evidence indicates that PD-L1 functions as a positive costimulatory molecule in intracellular infections.

The present disclosure provides anti-PD-L1 antibodies with high binding affinity for human PD-L1 protein, which can effectively block the interaction between PD-L1 and its receptor PD-1. It is also important in the examples that these anti-PD-L1 antibodies have been demonstrated to promote T cell immune responses and inhibit tumor growth. In contrast to known anti-PD-L1 antibodies, which bind to the immunoglobulin V domain of the extracellular portion of the PD-L1 protein, these antibodies bind to the immunoglobulin C domain, particularly to amino acid residues Y134, K162 and N183. To do. These anti-PD-L1 antibodies are useful for therapeutic purposes, such as the treatment of various types of cancers and infectious diseases, and can be used for diagnostic and prognostic purposes.

One embodiment of the disclosure provides an anti-PD-L1 antibody or antibody fragment, wherein the antibody or antibody fragment is specific for the immunoglobulin C (IgC) domain of human programmed cell death ligand 1 (PD-L1) protein. Can be combined with. In some embodiments, the IgC domain consists of amino acid residues 133-225. In some embodiments, the antibody or antibody fragment is capable of binding to at least one of amino acid residues Y134, K162 or N183 of the PD-L1 protein. In some embodiments, the antibody or antibody fragment is capable of binding to at least one of amino acid residues Y134, K162 and N183 of the PD-L1 protein. In some embodiments, the antibody or antibody fragment does not bind to the immunoglobulin V (IgV) domain of the PD-L1 protein and the IgV domain consists of amino acid residues 19-127.

One embodiment of the present disclosure provides an anti-PD-L1 antibody or antibody fragment, wherein the antibody or antibody fragment has specificity for human programmed cell death ligand 1 (PD-L1) protein and has a sequence identification number: VH CDR1 of 1; VH CDR2 of SEQ ID NO:2; VH CDR3 of SEQ ID NO:3; VL CDR1 of SEQ ID NO:4; VL CDR2 of SEQ ID NO:5; and VL CDR3 of SEQ ID NO:6. In some embodiments, the antibody or antibody fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or antibody fragment is an IgG, IgM, IgA, IgE or IgD isotype. In some embodiments, the isotype is IgG1, IgG2, IgG3 or IgG4. The antibody or antibody fragment is, but not limited to, a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, the antibody or antibody fragment is a humanized antibody.

Through mutagenesis, the disclosure further provides VH CDR3 (see, eg, antibodies A1, A2, C3, C4, C6, B1 and B6 in Examples 13-17) and VL CDR3 (eg, antibody B3 in Examples 13-17, Mutant hotspot residues in C4 and A3) were identified. Thus, the disclosure also provides antibodies that incorporate one or more mutations in these hotspots.

In some embodiments, the antibody or antibody fragment wherein the antibody or antibody fragment has specificity for human PD-L1 protein and comprises (a) SEQ ID NO:1, or SEQ ID NO:1. And comparing with VH CDR1, which comprises the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions, (b) SEQ ID NO: 116, or SEQ ID NO: 116, VH CDR2 containing the amino acid sequence of the variant of SEQ ID NO: 116 containing 1, 2 or 3 substitutions, deletions or insertions, (c) SEQ ID NO: 117 or 1, 2 compared to SEQ ID NO: 117 Alternatively, a VH CDR3 comprising the amino acid sequence of the variant of SEQ ID NO: 117 containing three substitutions, deletions or insertions, wherein the second amino acid residue is Leu, VH CDR3, (d) SEQ ID NO: 4, or A VL CDR1, comprising the amino acid sequence of a variant of SEQ ID NO:4 with 1, 2 or 3 substitutions, deletions or insertions compared to SEQ ID NO:4, (e) SEQ ID NO:5, or SEQ ID NO: VL CDR2 comprising the amino acid sequence of the variant of SEQ ID NO: 5 with 1, 2 or 3 substitutions, deletions or insertions compared to 5, and (f) SEQ ID NO: 6 or SEQ ID NO: 6 An antibody or antibody fragment comprising a VL CDR3 comprising the amino acid sequence of the variant of SEQ ID NO:6 with 1, 2 or 3 substitutions, deletions or insertions, is provided.

In one embodiment, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:116 and the VH CDR3 comprises the amino acid sequence of SEQ ID NO:117, VL CDR1 comprises the amino acid sequence of SEQ ID NO:4, said VL CDR2 comprises the amino acid sequence of SEQ ID NO:5 and said VL CDR3 comprises the amino acid sequence of SEQ ID NO:6.

In one embodiment, the antibody or antibody fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:149 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:150.

Also, in one embodiment, the antibody or antibody fragment has specificity for human PD-L1 protein and comprises (a) SEQ ID NO: 1 or SEQ ID NO: 1 And comparing with VH CDR1, which comprises the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions, (b) SEQ ID NO: 116, or SEQ ID NO: 116, VH CDR2 comprising the amino acid sequence of the variant of SEQ ID NO:116 with 1, 2 or 3 substitutions, deletions or insertions, (c) SEQ ID NO:3, or 1, 2 compared to SEQ ID NO:3 Or VH CDR3 containing the amino acid sequence of the variant of SEQ ID NO: 3 with 3 substitutions, deletions or insertions, (d) SEQ ID NO: 4 or 1, 2 or 3 compared to SEQ ID NO: 4 VL CDR1 containing the amino acid sequence of the variant of SEQ ID NO: 4 containing substitutions, deletions or insertions, (e) SEQ ID NO: 5 or 1, 2 or 3 substitutions, deletions compared to SEQ ID NO: 5 VL CDR2 containing the amino acid sequence of the variant of SEQ ID NO: 5 containing deletions or insertions, and (f) SEQ ID NO: 140, or 1, 2 or 3 substitutions, deletions as compared to SEQ ID NO: 140 Alternatively, at least (i) amino acid residue 4 of VL CDR3 is Ser, (ii) amino acid residue 5 of VL CDR3 is Asp, or (iii) ) An antibody or antibody fragment comprising VL CDR3 is provided wherein amino acid residue 6 of said VL CDR3 is Ala.

In one embodiment, VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, said VH CDR2 comprises the amino acid sequence of SEQ ID NO:116, said VH CDR3 comprises the amino acid sequence of SEQ ID NO:3, VL CDR1 comprises the amino acid sequence of SEQ ID NO:4, said VL CDR2 comprises the amino acid sequence of SEQ ID NO:5 and said VL CDR3 comprises the amino acid sequence of SEQ ID NO:140.

In one embodiment, the antibody or antibody fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:159 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:160.

In one embodiment, the antibody or antibody fragment wherein the antibody or antibody fragment has specificity for the human PD-L1 protein and comprises (a) SEQ ID NO:1 or as compared to SEQ ID NO:1. VH CDR1 comprising the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions, (b) SEQ ID NO: 116 or 1, compared to SEQ ID NO: 116, VH CDR2 comprising the amino acid sequence of the variant of SEQ ID NO:116 containing 2 or 3 substitutions, deletions or insertions, (c) SEQ ID NO:3, or 1, 2 or 3 compared to SEQ ID NO:3 VH CDR3 comprising the amino acid sequence of the variant of SEQ ID NO:3 containing one substitution, deletion or insertion, (d) SEQ ID NO:4, or 1, 2 or 3 substitutions compared to SEQ ID NO:4, VL CDR1, comprising the amino acid sequence of a variant of SEQ ID NO:4 with a deletion or insertion, (e) SEQ ID NO:5, or 1, 2 or 3 substitutions, deletions or as compared to SEQ ID NO:5 VL CDR2 comprising the amino acid sequence of the variant of SEQ ID NO: 5 with an insertion, and (f) SEQ ID NO: 6 or 1, 2 or 3 substitutions, deletions or insertions compared to SEQ ID NO: 6 An antibody or antibody fragment comprising a VL CDR3 comprising the amino acid sequence of the variant of SEQ ID NO: 6 having

In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:116, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO:3. The VL CDR1 includes the amino acid sequence of SEQ ID NO:4, the VL CDR2 includes the amino acid sequence of SEQ ID NO:5, and the VL CDR3 includes the amino acid sequence of SEQ ID NO:6.

In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:141 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:142.

Also provided in some embodiments are compositions comprising an antibody or antibody fragment of the present disclosure and a pharmaceutically acceptable carrier. Further, in some embodiments, an isolated cell is provided that comprises one or more polynucleotides encoding an antibody or antibody fragment of the present disclosure.

Treatment methods and uses are also provided. In one embodiment, there is provided a method of treating cancer or infection in a patient, comprising administering to a patient in need thereof an effective amount of an antibody or antibody fragment of the present disclosure. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck. It is selected from the group consisting of cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, ovarian cancer, renal cancer, malignant melanoma, prostate cancer, and thyroid cancer. In some embodiments, the cancer is bladder cancer, liver cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, It is selected from the group consisting of gastric cancer, esophageal cancer, ovarian cancer, renal cancer and small cell lung cancer. In some embodiments, the method further comprises administering a second cancer therapeutic agent to the patient. In some embodiments, the infection is a viral, bacterial, fungal, or parasitic infection.

In another embodiment, there is provided a method of treating cancer or infection in a patient in need thereof, comprising the step of: (a) treating cells in vitro with an antibody or antibody fragment of the present disclosure. And (b) administering the treated cells to a patient. In some embodiments, the method further comprises the step of isolating cells from the individual prior to step (a). In some embodiments, the cells are isolated from the patient. In some embodiments, the cells are isolated from a donor individual different from the patient. In some embodiments, the cells are T cells, non-limiting examples of which include tumor infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof.

Diagnostic methods and uses are also provided. In one embodiment, comprising contacting the sample with the antibody or antibody fragment under conditions in which the antibody or antibody fragment binds to PD-L1, and detecting binding that is indicative of PD-L1 expression in the sample. Provided is a method for detecting PD-L1 expression in a sample. In some embodiments, the sample comprises a tumor cell, tumor tissue, infected tissue or blood sample.

The antibodies and fragments of the present disclosure can be used to prepare bispecific antibodies. In one embodiment, a bispecific antibody is provided that comprises a fragment of the present disclosure and a second antigen-binding fragment that has specificity for molecules on immune cells. In some embodiments, the molecule is PD-1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA, selected from the group consisting of CD47 and CD73. In some embodiments, the fragment and the second fragment are each independently selected from Fab fragments, single chain variable fragments (scFv) or single domain antibodies. In some embodiments, the bispecific antibody further comprises an Fc fragment.

We show that HL1210-3 can bind to human PD-L1 with high affinity.

It is shown that HL1210-3 can efficiently inhibit the binding of human PD-L1 to human PD1.

It is shown that the HL1210-3 antibody can inhibit the binding of PD-1 to PD-L1 expressed on mammalian cells with high efficiency.

It is shown that the tested anti-PD-L1 antibody is capable of promoting a human T cell response.

The binding rate of HL1210-3 to recombinant PD-L1 is shown.

It is shown that all tested humanized antibodies have comparable binding potency to human PD-L1 as the chimeric antibody.

It is shown that all tested humanized antibodies can bind PD-L1 expressed on mammalian cells with as high efficiency as the chimeric antibody.

We show that the humanized antibody Hu1210-41 can bind to rhesus PD-L1 with low affinity and not to rat and mouse PD-L1.

Hu1210-41 antibody specifically binds only to B7-H1 (PD-L1) but not B7-DC, B7-1, B7-2, B7-H2, PD-1, CD28, CTLA4, ICOS and BTLA Show what you can do.

It is shown that Hu1210-41 can efficiently inhibit the binding of human PD-L1 to human PD1 and B7-1.

It is shown that Hu1210-41 can efficiently inhibit the binding of human PD-L1 to human PD1 and B7-1.

It is shown that Hu1210-8, Hu1210-9, Hu1210-16, Hu1210-17, Hu1210-21 and Hu1210-36 humanized antibodies can enhance IFNγ and IL-2 production in a mixed lymphocyte reaction in a dose-dependent manner.

It is shown that Hu1210-40, Hu1210-41 and Hu1210-17 humanized antibodies can enhance IFNγ production in a CMV recall assay in a dose-dependent manner.

It is shown that Hu1210-31 can inhibit tumor growth by 30% at 5 mg/kg in the HCC827-NSG xenograft model.

The Hu1210-41 antibody can inhibit tumor growth in a dose-dependent manner in the HCC827-NSG xenograft model, while tumor weight is also shown to be suppressed by the Hu1210-41 antibody in a dose-dependent manner.

Mean binding values are plotted for each PD-L1 variant as a function of expression (control: anti-PD-L1 mAb reactivity).

The positions of Y134, K162 and N183, which are residues involved in binding to the anti-PD-L1Hu1210-41 antibody, are shown (in sphere).

The S60R mutant is compared to the parent antibody Hu1210-41 in terms of binding efficiency to PD-L1 expressed in mammalian cells.

The result of the binding assay (to human PD-L1) about the obtained antibody is shown.

Antibody B6 is shown to bind to PD-L1 expressed in mammalian cells with higher efficiency compared to the parent antibody and Tecentriq® (atezolizumab).

Figure 7 shows the effect of antibodies on IL2 production in Jurkat cells where B6 shows higher potency.

Figure 7 shows the in vitro activity of antibodies promoting IFNγ production in a mixed lymphocyte environment.

Figure 7 shows in vivo activity of antibodies that inhibit tumor growth.

[Definition]
Note that the entity "1" or "one" refers to one or more entities. For example, "one antibody" should be understood to mean one or more antibodies. Therefore, the terms "1" (or "one"), "one or more", and "at least one" can be used interchangeably herein.

The term "polypeptide" as used herein is intended to encompass a single "polypeptide" and multiple "polypeptides", also known as amide bonds (also known as peptide bonds). ) Refers to a molecule composed of monomers (amino acids) linearly linked by each other. The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a particular length of product. Thus, a peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other phrase used to refer to a chain of two or more amino acids or multiple chains refers to " Within the definition of "polypeptide," the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The phrase "polypeptide" also includes, but is not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, protein cleavage, or modification with non-naturally occurring amino acids. Is intended to refer to the product of post-expression modifications of the polypeptide. A polypeptide may be derived from a natural biological source or may be produced by recombinant technology, but is not necessarily translated from the designated nucleic acid sequence. The polypeptide may be produced in any manner, including chemical synthesis.

As used herein, the term “isolated” when used in reference to a cell, nucleic acid such as DNA or RNA, is a molecule that is isolated from other DNA or RNA, respectively, present in the natural source of the macromolecule. Refers to. The term "isolated" as used herein, refers to cellular material, viral material or media when produced by recombinant DNA techniques, or precursor chemicals when chemically synthesized. Alternatively, it may refer to a nucleic acid or peptide substantially free of other chemical substances. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that do not naturally occur as fragments and which may not be found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides isolated from other cellular proteins or tissues. Isolated polypeptide is meant to include both purified and recombinant polypeptides.

As used herein, the term "recombinant" when referring to a polypeptide or polynucleotide, refers to non-naturally occurring forms of the polypeptide or polynucleotide, non-limiting examples of which include: It can be made by combining polynucleotides or polypeptides that would not normally occur together.

“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that may be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "heterologous" sequence shares less than 40% identity (preferably less than 25% identity) with one of the disclosed sequences.

A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) and another sequence in a specific proportion (eg, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, Having 98% or 99% sequence identity means that when aligned, that percentage of bases (or amino acids) is identical in the comparison of the two sequences. This alignment and the percentage homology or sequence identity is described, for example, in Ausubel et al. eds. (2007) Can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology. Preferably, default parameters are used for the alignment. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters. Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. A biologically equivalent polynucleotide is one which encodes a polypeptide having the indicated percentage homology as set forth above, and having the same or similar biological activity.

The phrase "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has some homology or sequence identity with the nucleotide sequence of the nucleic acid or its complementary sequence. The double-stranded nucleic acid homolog is intended to include a nucleic acid having a nucleotide sequence having a certain degree of homology, or a complementary sequence thereof. In one aspect, a homologue of a nucleic acid is capable of hybridizing to the nucleic acid or its complementary sequence. Similarly, "equivalent polypeptide" refers to a polypeptide that has some homology or sequence identity with the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In some embodiments, equivalent polypeptides or polynucleotides have 1, 2, 3, 4 or 5 additions, deletions, substitutions and combinations when compared to the reference polypeptide or polynucleotide. In some embodiments, the equivalent sequences retain the activity (eg, epitope binding) or structure (eg, salt bridge) of the reference sequence.

Hybridization reactions can be performed under conditions of different "stringency". Generally, the low stringency hybridization reaction is carried out at about 40° C. in about 10 times the SSC solution, or a solution of comparable ionic strength/temperature. Hybridizations of moderate stringency are typically performed in about 6X SSC at about 50°C. High stringency hybridization reactions are generally performed at about 60° C. in about 1×SSC. Hybridization reactions can also be performed under "physiological conditions" which are well known to those of skill in the art. Non-limiting examples of physiological conditions are temperature, ions, strength, pH, and Mg ²⁺ concentration normally found in cells.

A polynucleotide has a specific sequence of four nucleotide bases, namely adenine (A), cytosine (C), guanine (G), thymine (T) (when the polynucleotide is RNA, uracil (U) instead of thymine (U). )). Thus, the phrase "polynucleotide sequence" is a letter representation of a polynucleotide molecule. This textual representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term "polymorphism" refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene that has at least two different forms, ie, two different nucleotide sequences, is called a "polymorphic region of the gene." Polymorphic regions may be single nucleotides and differ in identity at different alleles.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide can have any three-dimensional structure and perform any known or unknown function. The following are non-limiting examples of polynucleotides. Gene or gene fragment (eg, probe, primer, EST or SAGE tag), exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotide, branched type Polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can include modified nucleotides such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if any, can be imparted before or after the construction of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by ligation to a labeling moiety. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise stated or required, the polynucleotide of any embodiment of the present disclosure is in the double-stranded form and is known to form the double-stranded form, or Predicted and each of the two complementary single-stranded forms.

The term "code" as applied to a polynucleotide refers to the transcription and/or translation of the polypeptide and/or when the polynucleotide is in its native form or when manipulated by methods well known to those of skill in the art. Alternatively, it refers to a polynucleotide that is said to "encode" a polypeptide if it is able to produce mRNA for the fragment. The antisense strand is the complementary strand of such a nucleic acid, from which the coding sequence can be deduced.

As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. Antibodies may be whole antibodies and any antigen binding fragment or single chains thereof. Thus, the term "antibody" includes any protein or peptide that comprises a molecule that comprises at least a portion of an immunoglobulin molecule that has the biological activity of binding an antigen. Such examples include, but are not limited to, heavy chain or light chain complementarity determining regions (CDRs) or their ligand binding portions, heavy chain or light chain variable regions, heavy chain or light chain constant regions, frames. The work (FR) region, or any portion thereof, or at least one portion of the binding protein is included.

As used herein, the phrase "antibody fragment" or "antigen-binding fragment" refers to F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, and the like. Is part of the antibody. Regardless of structure, an antibody fragment binds to the same antigen recognized by intact antibodies. The phrase "antibody fragment" includes aptamers, spigelmers and bispecific antibodies. The term "antibody fragment" also includes synthetic or recombinant proteins that function like antibodies by binding to a specific antigen to form a complex.

"Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable region of an immunoglobulin heavy chain ( _VH ) and light chain ( _VL ). In some embodiments, this region binds a short linker peptide of 10 to about 25 amino acids. The linker may be rich in glycine for flexibility and serine or threonine for solubility and may be attached to either the N-terminus of _VH or the C-terminus of _VL (and vice versa). To). This protein has the constant region removed and a linker introduced, but retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in US Pat. No. 5,892,019.

The term antibody includes various broad classes of polypeptides that are biochemically distinct. Those skilled in the art will recognize that heavy chains are gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and some subclasses therein (eg, γ1-γ4). You will understand that it is classified into. The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. It is known that immunoglobulin subclasses (isotypes) such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , and IgG ₅ have distinct features and impart functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those of skill in the art upon reference to this disclosure and are therefore within the scope of this disclosure. It is clear that all immunoglobulin classes are within the scope of the present disclosure, and the following description generally relates to the IgG class of immunoglobulin molecules. In the context of IgG, a standard immunoglobulin molecule is two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. including. The four chains are typically linked in the form of a "Y" by a disulfide bond, the light chain pairing with the heavy chain starting at the open portion of the "Y" and contiguous throughout the variable region. ing.

Antibodies, antigen binding polypeptides, variants or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, humanized, humanized, primatized or chimeric antibodies, single chain antibodies. , An epitope-binding fragment, eg, Fab, Fab′ and F(ab′) ₂ , Fd, Fvs, single chain Fv (scFv), single chain antibody, disulfide linked Fv (sdFv), VK or VH domain , Fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). The immunoglobulins or antibody molecules of the present disclosure can be of any type (eg, IgG, IgE, IgM, IgD, IgA, IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or immunoglobulin. It can be a subclass of molecules.

Light chains are classified as either kappa or lambda (K, λ). Each heavy chain class can bind to either kappa or lambda light chains. Generally, the light and heavy chains are covalently linked to each other and when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell, the "tail" portions of the two heavy chains are covalently linked. They are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence runs from the N-terminus at the Y-shaped branch end to the C-terminus at the bottom of each chain.

Both light and heavy chains are divided into regions of structural and functional homology. The terms "steady" and "variable" are used for function. In this context, it is to be understood that the variable domains of both the light chain (VK) and heavy chain (VH) parts determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and heavy chain (CH1, CH2 or CH3) have important biological properties such as secretion, transplacental mobility, Fc receptor binding, complementary binding and the like. Is given. By convention, the numbering of the constant region domains increases with distance from the antigen binding site or amino terminus of the antibody. The N-terminal and C-terminal portions in the variable region are the constant regions and the CH3 and CK domains actually contain the carboxy-termini of the heavy and light chains, respectively.

As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK and VH domains of an antibody, or a subset of complementarity determining regions (CDRs), combine to form variable regions that define a three-dimensional antigen binding site. This four-element antibody structure forms the antigen binding site present at the end of each arm of Y. More specifically, the antigen binding site is defined by three CDRs in each of the VH and VK chains (ie CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). Defined. In some cases, for example, in certain immunoglobulin molecules from the camelid family or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule consists of only the heavy chain and no light chain. It is possible. For example, Hamers-Casterman et al. , Nature 363:446-448 (1993).

In naturally-occurring antibodies, the six "complementarity determining regions" or CDRs present in each antigen binding domain are short, non-contiguous sequences of amino acids, which when the antibody adopts a three-dimensional organization in an aqueous environment. Specifically positioned to form an antigen binding domain. The remaining amino acids in the antigen binding domain are called the "framework" regions and exhibit smaller intermolecular variability. The framework regions mainly take the form of β-sheets, and the CDRs form loops that connect the β-sheet structures and, in some cases, form part of the β-sheet structure. Thus, the framework regions function to form the scaffold for orienting the CDRs by non-covalent interactions between the chains. The antigen binding domain formed by the positioned CDRs defines a surface complementary to an epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to cognate epitopes. The amino acids that make up the CDR and framework regions, respectively, have been precisely defined ("Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Health and Health and Health and Health and Health and Health and Health and Health and Health and Health and Health (and Health)). 1983), and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)), any heavy or light chain variable region can be readily identified by one of skill in the art. it can.

Where there is more than one definition of a phrase used and/or accepted in the art, the definition of the phrase as used herein has all such meanings, unless expressly stated otherwise. Is intended to be included. A specific example is the use of the phrase "complementarity determining region"("CDR") to describe the discontinuous antigen binding sites found in the variable regions of both heavy and light chain polypeptides. is there. This particular region is described by Kabat et al. , U. S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983), and Chothia et al. , J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety. The definition of CDR by Kabat and Chothia includes a subset of overlapping amino acid residues when compared to each other. Notwithstanding the above, applying any definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the terms defined and used herein. Suitable amino acid residues, including the CDRs defined by each of the above cited references, are set forth in the table below for comparison. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residue constitutes a particular CDR by considering the variable region amino acid sequence of the antibody.

Kabat et al also defined a numbering system for variable domain sequences applied to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without resorting to any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to Kabat et al. , U. S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

In addition to the table above, the Kabat numbering system describes CDR regions as follows. CDR-H1 begins at about the 31st amino acid (ie, about 9 residues after the first cysteine residue), contains about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue after the end of CDR-H1 and contains approximately 16-19 amino acids, ending at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2 and contains 3-25 amino acids and ends in the sequence W-G-X-G, where X is any amino acid. , CDR-L1 starts at about the 24th residue (ie, after the cysteine residue), contains about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 starts at approximately the 33rd residue after the end of CDR-L2 (ie, after the cysteine residue) and contains approximately 7-11 residues, and contains F or W-G-X-G X is an arbitrary amino acid.

The antibodies disclosed herein can be of any animal origin, including birds and mammals. Preferably, the antibody is a human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody. In another embodiment, the variable region may originate from a condricthoid (eg from a shark).

As used herein, the phrase "heavy chain constant region" includes amino acid sequences derived from immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region can be a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. At least one is included. For example, an antigen binding polypeptide for use in the present disclosure includes (a) a CH1 domain-containing polypeptide chain, (b) a CH1 domain-containing polypeptide chain, (c) at least a portion of a hinge domain and a CH2 domain, (D) CH1 domain- and CH3 domain-containing polypeptide chain, (e) CH1 domain, at least part of hinge domain, and CH3 domain-containing polypeptide chain, or (f) CH1 domain, at least one of hinge domain Part, a CH2 domain, and a CH3 domain-containing polypeptide chain. In another embodiment, a polypeptide of the present disclosure comprises a polypeptide chain that comprises a CH3 domain. Furthermore, the antibodies used in the present disclosure can be lacking at least a portion of the CH2 domain (eg, all or a portion of the CH2 domain). As explained above, those skilled in the art will appreciate that the heavy chain constant region may be modified so that it has a different amino acid sequence than the naturally occurring immunoglobulin molecule.

The heavy chain constant region of the antibodies disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain from an IgG ₁ molecule and a hinge region from an IgG ₃ molecule. In another example, the heavy chain constant region can include a hinge region, which is derived in part from an IgG ₁ molecule and in part from an IgG ₃ molecule. In another example, the heavy chain portion can include a chimeric hinge that is partially derived from an IgG ₁ molecule and partially derived from an IgG ₄ molecule.

As used herein, the phrase "light chain constant region" includes amino acid sequences derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

A "light chain-heavy chain pair" refers to a pair of light and heavy chains that is capable of forming a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

As mentioned above, the subunit structure and three-dimensional organization of the constant region of various immunoglobulin classes is well known. As used herein, the phrase "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain and the phrase "CH1 domain" refers to the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. including. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of immunoglobulin heavy chain molecules.

The term "CH2 domain" as used herein includes, for example, the portion of the heavy chain molecule that extends from about residue 244 to residue 360 of the antibody using conventional numbering schemes. (Residues 244 to 360 for the Kabat numbering system, residues 231 to 340 for the EU numbering system, Kabat et al., U.S. Dept. reference). The CH2 domain is unique in that it is not closely paired with another domain. Rather, the two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the complete native IgG molecule. It is also well established that the CH3 domain extends from the CH2 domain to the C-terminus of IgG molecules and contains approximately 108 residues.

As used herein, the phrase "hinge region" includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three distinct domains, an upper, a central and a lower hinge domain (Roux et al., J. Immunol 161:4083 (1998)).

The term "disulfide bond" as used herein includes covalent bonds formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond and the two heavy chains are 239 and 242 when using the Kabat numbering system (position 226 or 229 for the EU numbering system). ) Are linked by two disulfide bonds at the positions corresponding to.

As used herein, the phrase "chimeric antibody" refers to an immunoreactive region or site obtained from, or derived from, a first species, and a constant region (according to the present disclosure intact). , Partial, or modified) can be obtained from the second species. In certain embodiments, the target binding region or site is from a non-human source (eg mouse or primate) and the constant region is human.

As used herein, "humanization rate" determines the difference in the number of framework amino acids (ie, the non-CDR differences) between the humanized and germline domains, and that number is the total amino acid. Calculated by subtracting from the number, dividing it by the total number of amino acids, and multiplying by 100.

"Specifically binding" or "having specificity" generally means that the antibody binds to the epitope through the antigen binding domain, and that binding involves some complementarity between the antigen binding domain and the epitope. Means to accompany. By this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope via its antigen binding domain more easily than it binds to a random unrelated epitope. The term "specificity" is used herein to recognize the relative affinity of a particular antibody for binding a particular epitope. For example, antibody "A" may be considered to have a higher specificity for any epitope than antibody "B", or antibody "A" may be more specific than the related epitope "D". Can be said to bind to an epitope.

The term "treatment" or "treating" as used herein refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent unwanted physiological changes or disorders, such as the progression of cancer. Slow down (easy). Beneficial or desirable clinical outcomes include, but are not limited to, detectable, diminished symptoms, diminished severity of disease, stabilization of disease (ie, no exacerbation), and progression of disease. Includes delay or blunting, amelioration or alleviation of a medical condition, and remission (whether complete or partial). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in whom the condition or disorder is to be prevented.

"Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, especially a mammalian subject for whom diagnosis, prognosis or treatment is desirable. Mammalian subjects include humans, livestock animals, agricultural animals, or dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, zoos, sports, or pet animals. ..

As used herein, phrases such as “in a patient in need of treatment” or “subject in need of treatment” are used in, for example, detection, diagnostic procedures, and/or treatment. Including subjects, such as mammalian subjects, who would benefit from the administration of the antibodies or compositions of.

[Anti-PD-L1 antibody]
The present disclosure provides anti-PD-L1 antibodies with high affinity for human PD-L1 protein. The tested antibodies show strong binding and inhibitory activity and are therefore useful in therapeutic and diagnostic applications.

The PD-L1 protein is a 40 kDa type 1 cell transmembrane protein. The extracellular portion contains an N-terminal immunoglobulin V (IgV) domain (amino acids 19-127) and a C-terminal immunoglobulin C (IgC) domain (amino acids 133-225). PD-1 and PD-L1 interact through the conserved anterior and lateral sides of the IgV domain, like the IgV domain of an antibody and the T cell receptor. Of course, all current anti-PD-L1 antibodies bind to the IgV domain which can interfere with the binding between PD-1 and PD-L1. Thus, antibodies, such as many of the ones disclosed herein, that bind to the IgC domain of the PD-L1 protein can still effectively, and perhaps even more, inhibit PD-L1 with even further improved therapeutic efficacy. The discovery of the present disclosure that results in is surprising and unexpected.

Accordingly, one embodiment of the present disclosure provides an anti-PD-L1 antibody or antibody fragment, wherein the antibody or antibody fragment is in the immunoglobulin C (IgC) domain of human programmed cell death ligand 1 (PD-L1) protein. Can bind specifically. In some embodiments, the IgC domain consists of amino acid residues 133-225.

In some embodiments, the antibody or antibody fragment is capable of binding to at least one of amino acid residues Y134, K162 or N183 of the PD-L1 protein. In some embodiments, the antibody or antibody fragment is capable of binding at least two of amino acid residues Y134, K162 or N183 of the PD-L1 protein. In some embodiments, the antibody or antibody fragment is capable of binding to at least one of amino acid residues Y134, K162 and N183 of the PD-L1 protein. In some embodiments, the antibody or antibody fragment does not bind to the immunoglobulin V (IgV) domain of the PD-L1 protein and the IgV domain consists of amino acid residues 19-127.

According to one embodiment of the disclosure there is provided an antibody comprising heavy and light chain variable domains having CDR regions as defined in SEQ ID NOs: 1-6.
[Table 1 Sequence of CDR region]

As shown in the experimental examples, antibodies containing these CDR regions, whether mouse, humanized or chimeric, have potent PD-L1 binding and inhibitory activity. Further computer modeling has shown that certain residues in the CDRs can be modified to preserve or improve the properties of the antibody. Such residues are called "hot spots" and are underlined in Table 1. In some embodiments, anti-PD-L1 antibodies of the present disclosure comprise VH and VL CDRs with 1, 2, or 3 further modifications, as listed in Table 1. Such modifications can be amino acid additions, deletions, or substitutions.

In some embodiments, the modification is a substitution at only one hotspot position in each of the CDRs. In some embodiments, the modification is a substitution at 1, 2 or 3 such hotspot positions. In one embodiment, the modification is a substitution at one of the hotspot positions. In some embodiments, such substitutions are conservative substitutions.

A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains as defined in the art include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar. Side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains ( Examples include threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue of the same side chain family. In another embodiment, a chain of amino acids can be replaced with a structurally similar chain that differs in the order and/or composition of side chain family members.

Non-limiting examples of conservative amino acid substitutions are provided in the table below. Here, a similarity score of 0 or higher indicates a conservative substitution between two amino acids.
[Table 2: Amino acid similarity matrix]
[Table 3. Conservative amino acid substitution]

Specific examples of CDRs with suitable substitutions are provided in SEQ ID NOs: 61-111 of Example 11. Thus, in some embodiments, an antibody of the present disclosure comprises a VH CDR1 of any one of SEQ ID NO: 1 or 61-67. In some embodiments, the antibody of the present disclosure comprises a VH CDR2 of any one of SEQ ID NO:2 or 68-77. In some embodiments, the antibody of the present disclosure comprises a VH CDR3 of any one of SEQ ID NO: 1 or 78-90. In some embodiments, the antibody of the present disclosure comprises a VL CDR1 of any one of SEQ ID NO:4 or 91-92. In some embodiments, the antibody of the present disclosure comprises a VL CDR2 of any one of SEQ ID NO:5 or 93-105. In some embodiments, the antibody of the present disclosure comprises a VL CDR3 of any one of SEQ ID NO:6 or 106-110.

In some embodiments, the antibody or antibody fragment comprises only one, only two, or only three of the above substitutions. In some embodiments, the antibody or antibody fragment comprises a VH CDR1 of any one of SEQ ID NO:1 or SEQ ID NOs:61-67, a VH CDR2 of SEQ ID NO:2, and a VH CDR3 of SEQ ID NO:3. VL CDR1 having sequence identification number 4, VL CDR2 having sequence identification number 5, and VL CDR3 having sequence identification number 6.

In some embodiments, the antibody or antibody fragment comprises VH CDR1 of SEQ ID NO:1, VH CDR2 of any one of SEQ ID NO:2 or SEQ ID NO:68-77, and VH CDR3 of SEQ ID NO:3. VL CDR1 having sequence identification number 4, VL CDR2 having sequence identification number 5, and VL CDR3 having sequence identification number 6.

In some embodiments, the antibody or antibody fragment comprises VH CDR1 of SEQ ID NO:1, VH CDR2 of SEQ ID NO:2, and VH CDR3 of any one of SEQ ID NO:3 or SEQ ID NO:78-90. VL CDR1 having sequence identification number 4, VL CDR2 having sequence identification number 5, and VL CDR3 having sequence identification number 6.

In some embodiments, the antibody or antibody fragment comprises VH CDR1 of SEQ ID NO:1, VH CDR2 of SEQ ID NO:2, VH CDR3 of SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:91- VL CDR1 of any one of 92, VL CDR2 of SEQ ID NO:5, and VL CDR3 of SEQ ID NO:6.

In some embodiments, the antibody or antibody fragment comprises VH CDR1 of SEQ ID NO:1, VH CDR2 of SEQ ID NO:2, VH CDR3 of SEQ ID NO:3, VL CDR1 of SEQ ID NO:4, and VL CDR2 of any one of SEQ ID NO:5 or SEQ ID NO:93-105 and VL CDR3 of SEQ ID NO:6.

In some embodiments, the antibody or antibody fragment comprises VH CDR1 of SEQ ID NO:1, VH CDR2 of SEQ ID NO:2, VH CDR3 of SEQ ID NO:3, VL CDR1 of SEQ ID NO:4, and VL CDR2 with identification number 5 and VL CDR3 with any one of SEQ ID NO:6 or SEQ ID NO:106-111.

Non-limiting examples of VHs are provided in SEQ ID NOs:7-26 and 113, of which SEQ ID NO:113 is a mouse VH and SEQ ID NOs:7-26 are humanized VHs. Furthermore, of the humanized VH, SEQ ID NOs: 9-15, 17-21 and 23-26 contain one or more back mutations to the mouse version. Similarly, non-limiting examples of VL(VK) are provided in SEQ ID NOs:27-33. SEQ ID NOs: 28 and 30 are CDR-grafted humanized sequences originally obtained, as shown in the examples. SEQ ID NOs: 29 and 31-33 are humanized VLs with back mutations.

Back mutations have been shown to be useful for retaining certain features of anti-PD-L1 antibodies. Thus, in some embodiments, anti-PD-L1 antibodies of the present disclosure, particularly humanized or humanized antibodies, include one or more of the back mutations. In some embodiments, the VH backmutation (ie, includes an amino acid at a designated position) is performed by Kabat numbering, (a) Ser at position 44, (b) Ala at position 49, (c) position 53. Ala, (d) Ile at position 91, (e) Glu at position 1, (f) Val at position 37, (g) Thr at position 40, (h) Val at position 53, and (i) Glu at position 54. , (J) Asn at position 77, (k) Arg at position 94, and (l) Thr at position 108, and one or more selected from combinations thereof. In some embodiments, the backmutation is due to Kabat numbering: (a) Ser at position 44, (b) Ala at position 49, (c) Ala at position 53, and/or (d) at position 91. Selected from Ile and combinations thereof.

In some embodiments, the VL backmutation is due to Kabat numbering: (a) Ser at position 22, (b) Gln at position 42, (c) Ser at position 43, (d) Asp at position 60, and , (E) Thr at position 63, and one or more selected from combinations thereof.

In some embodiments, an anti-PD-L1 antibody of the present disclosure comprises a VH of SEQ ID NOs:7-26, a VL of SEQ ID NOs:27-33, or respective bioequivalents. VH or VL bioequivalents are sequences containing the specified amino acids with overall 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. A bioequivalent of SEQ ID NO:20, for example, has overall 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO:20, but the CDR (sequence VH carrying the identification numbers 1-6 or variants thereof and optionally carrying one or more or all of the back mutations. In one embodiment, VH has the amino acid sequence of SEQ ID NO:20 and VL has the amino acid sequence of SEQ ID NO:28.

[Further improved PD-L1 antibody]
Through random mutagenesis with controlled mutagenesis, Examples 13-17, in particular, show hotspot residue in CDR3 of both heavy chain (eg, B6, C3, C6 and A1) and light chain (eg, A3) variable regions. The number of groups could be identified (see Tables 14 and 15). Mutagenesis was performed on a template antibody derived from Hu1210-41 (the template antibody WT has an S60R (Kabat numbering) substitution in the heavy chain CDR2, as shown in the footnote of Table 14). Also, Hu1210-41 contains a G53A substitution (see SEQ ID NO:20) in VH CDR2, as compared to the chimeric antibody. Of the mutant antibodies tested, antibody B6 showed greatly improved binding affinity and biological activity for human PD-L1.

Thus, in one embodiment, there are provided antibodies and antigen binding fragments comprising the following CDRs (from the S60R variant), and variants thereof.

Thus, in one embodiment, there are provided antibodies and antigen binding fragments comprising the following CDRs (from B6) and variants thereof.

In one embodiment, the antibody or antibody fragment, wherein the antibody or antibody fragment has specificity for human PD-L1 protein, wherein (a) SEQ ID NO: 1 or, relative to SEQ ID NO: 1, VH CDR1 comprising the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions, (b) SEQ ID NO: 116 or 1, compared to SEQ ID NO: 116, VH CDR2 comprising the amino acid sequence of the variant of SEQ ID NO:116 containing 2 or 3 substitutions, deletions or insertions, (c) SEQ ID NO:3, or 1, 2 or 3 compared to SEQ ID NO:3 VH CDR3 comprising the amino acid sequence of the variant of SEQ ID NO:3 containing one substitution, deletion or insertion, wherein the second amino acid residue is Leu, (d) SEQ ID NO:4, or SEQ ID NO: VL CDR1 comprising the amino acid sequence of the variant of SEQ ID NO: 4 with 1, 2 or 3 substitutions, deletions or insertions compared to SEQ ID NO: 4, (e) SEQ ID NO: 5 or SEQ ID NO: 5 In comparison, a VL CDR2 comprising the amino acid sequence of a variant of SEQ ID NO:5 having 1, 2 or 3 substitutions, deletions or insertions, and (f) SEQ ID NO:6 or compared to SEQ ID NO:6. Thus, there is provided an antibody or antibody fragment comprising a VL CDR3 that comprises the amino acid sequence of the variant of SEQ ID NO:6 with 1, 2 or 3 substitutions, deletions or insertions.

In one embodiment, the antibody or antibody fragment, wherein the antibody or antibody fragment has specificity for human PD-L1 protein, wherein (a) SEQ ID NO: 1 or, relative to SEQ ID NO: 1, VH CDR1 comprising the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions, (b) SEQ ID NO: 116 or 1, compared to SEQ ID NO: 116, VH CDR2 comprising the amino acid sequence of the variant of SEQ ID NO: 116 containing 2 or 3 substitutions, deletions or insertions, (c) SEQ ID NO: 117, or 1, 2 or 3 compared to SEQ ID NO: 117 VH CDR3 comprising the amino acid sequence of the variant of SEQ ID NO: 117 containing one substitution, deletion or insertion, wherein the second amino acid residue is Leu, (D) SEQ ID NO: 4 or SEQ ID NO: VL CDR1 comprising the amino acid sequence of the variant of SEQ ID NO: 4 with 1, 2 or 3 substitutions, deletions or insertions compared to SEQ ID NO: 4, (e) SEQ ID NO: 5 or SEQ ID NO: 5 In comparison, a VL CDR2 comprising the amino acid sequence of a variant of SEQ ID NO:5 having 1, 2 or 3 substitutions, deletions or insertions, and (f) SEQ ID NO:6 or compared to SEQ ID NO:6. Thus, there is provided an antibody or antibody fragment comprising a VL CDR3 that comprises the amino acid sequence of the variant of SEQ ID NO:6 with 1, 2 or 3 substitutions, deletions or insertions.

Examples of variants of SEQ ID NO:1, such as SEQ ID NO:61-67, have one amino acid substitution at one of amino acid residues 1, 2 and 5.

Examples of variants of SEQ ID NO:116 have one or more amino acid substitutions, such as SEQ ID NO:118-127, 2 and 68-77. In some embodiments, the variant is SEQ ID NO: 118-127.

In some embodiments, the third amino acid residue of the VH CDR3 variant is Pro. In some embodiments, the fourth amino acid residue of the VH CDR3 variant is Trp.

Examples of variants of SEQ ID NO:3, such as SEQ ID NO:78-90, have one or more amino acid substitutions at amino acid residues 1-6.

Examples of variants of SEQ ID NO: 117, such as SEQ ID NO: 128-139, have one or more amino acid substitutions at amino acid residues 1, 5 and 6.

In some embodiments, the variant of SEQ ID NO:4, such as SEQ ID NO:91-92, has a single amino acid substitution at amino acid residue 3.

In some embodiments, the variant of SEQ ID NO:5, such as SEQ ID NO:93-105, has a single amino acid substitution at one of amino acid residues 1-6.

Examples of variants of SEQ ID NO: 6, such as SEQ ID NO: 106-111, have one amino acid substitution at one of amino acid residues 1 and 2. Another example variant is SEQ ID NO:140.

Mutant A3 with a substitution at 3 residues in VL CDR3 also shows excellent binding affinity for human PD-L1. Thus, in one embodiment, antibodies and antigen binding fragments comprising the following CDRs and variants thereof are provided.

Therefore, in one embodiment, the antibody or antibody fragment has specificity for human PD-L1 protein and comprises (a) SEQ ID NO: 1 or compared to SEQ ID NO: 1. And comparing with VH CDR1, which comprises the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions, (b) SEQ ID NO: 116, or SEQ ID NO: 116, VH CDR2 comprising the amino acid sequence of the variant of SEQ ID NO:116 with 1, 2 or 3 substitutions, deletions or insertions, (c) SEQ ID NO:3, or 1, 2 compared to SEQ ID NO:3 Or VH CDR3 containing the amino acid sequence of the variant of SEQ ID NO: 3 with 3 substitutions, deletions or insertions, (d) SEQ ID NO: 4 or 1, 2 or 3 compared to SEQ ID NO: 4 VL CDR1 containing the amino acid sequence of the variant of SEQ ID NO: 4 containing substitutions, deletions or insertions, (e) SEQ ID NO: 5 or 1, 2 or 3 substitutions, deletions compared to SEQ ID NO: 5 VL CDR2 containing the amino acid sequence of the variant of SEQ ID NO: 5 containing deletions or insertions, and (f) SEQ ID NO: 140, or 1, 2 or 3 substitutions, deletions as compared to SEQ ID NO: 140 Or at least (i) the amino acid residue 4 of the VL CDR3 is Ser, (ii) the amino acid residue 5 of the VL CDR3 is Asp, or (iii) ) An antibody or antibody fragment comprising VL CDR3 is provided wherein amino acid residue 6 of said VL CDR3 is Ala.

Examples of variants of SEQ ID NO:1, such as SEQ ID NO:61-67, have one amino acid substitution at one of amino acid residues 1, 2 and 5.

Examples of variants of SEQ ID NO:116 have one or more amino acid substitutions, such as SEQ ID NO:118-127, 2 and 68-77.

Examples of variants of SEQ ID NO:3, such as SEQ ID NO:117 and 128-139, have one or more amino acid substitutions.

Examples of variants of SEQ ID NO:4, such as SEQ ID NO:91-92, have a single amino acid substitution at amino acid residue 3.

Examples of variants of SEQ ID NO:5, such as SEQ ID NO:93-105, have one amino acid substitution at one of amino acid residues 1-6.

In some embodiments, amino acid residue 4 of the VL CDR3 variant is Ser. In some embodiments, amino acid residue 5 of the VL CDR3 variant is Asp. In some embodiments, amino acid residue 6 of the VL CDR3 variant is Ala. Examples of variants of SEQ ID NO:140, such as SEQ ID NO:161-166, have one amino acid substitution at one of amino acid residues 1 and 2.

Examples of antibodies, or antigen binding fragments thereof, obtained from mutagenesis studies have the heavy and light chain variable regions provided in Table 15. In one embodiment, the heavy chain variable region comprises SEQ ID NO:141 and the light chain variable region comprises SEQ ID NO:142. In one embodiment, the heavy chain variable region comprises SEQ ID NO:143 and the light chain variable region comprises SEQ ID NO:144. In one embodiment, the heavy chain variable region comprises SEQ ID NO:145 and the light chain variable region comprises SEQ ID NO:146. In one embodiment, the heavy chain variable region comprises SEQ ID NO:147 and the light chain variable region comprises SEQ ID NO:148. In one embodiment, the heavy chain variable region comprises SEQ ID NO:149 and the light chain variable region comprises SEQ ID NO:150. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 151 and the light chain variable region comprises SEQ ID NO: 152. In one embodiment, the heavy chain variable region comprises SEQ ID NO:153 and the light chain variable region comprises SEQ ID NO:154. In one embodiment, the heavy chain variable region comprises SEQ ID NO:155 and the light chain variable region comprises SEQ ID NO:156. In one embodiment, the heavy chain variable region comprises SEQ ID NO:157 and the light chain variable region comprises SEQ ID NO:158. In one embodiment, the heavy chain variable region comprises SEQ ID NO:159 and the light chain variable region comprises SEQ ID NO:160.

One of ordinary skill in the art will also appreciate that the antibodies disclosed herein may be modified to vary in amino acid sequence as compared to the naturally-occurring binding polypeptide from which they are derived. Will. For example, the polypeptide or amino acid sequences from the designated proteins can be similar, eg, have a certain percent identity to the starting sequence, eg, 60% to the starting sequence, It can be 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical.

In certain embodiments, the antibody comprises an amino acid sequence, or one or more moieties, that is not normally associated with the antibody. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure can include flexible linker sequences or can be modified to add functional groups (eg, PEG, drugs, toxins, or labels).

Antibodies, variants, or derivatives thereof of the present disclosure are modified derivatives, i.e., modified by covalently attaching any type of molecule to the antibody such that binding of the antibody to the epitope is not covalently prevented. Included derivatives. For example, but not limited to, antibodies can be, for example, glycosylated, acetylated, PEGylated, phosphorylated, amidated, derivatized with known protecting/blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Can be modified by Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the antibody may contain one or more non-conventional amino acids.

In some embodiments, the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, drug, or PEG.

Antibodies are therapeutic agents that may include detectable labels such as radioactive labels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic agents, photoactive diagnostic agents, cytotoxic agents that may be drugs or poisons, It can be attached or fused to sonic contrast agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

The antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescently labeled antigen binding polypeptide is then determined by detecting the presence of luminescence that occurs during the course of the chemimetric reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

Antibodies can also be detectably labeled using fluorescent emitting metals such as ¹⁵² Eu or others of the lanthanide series. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for coupling various moieties to antibodies are known. For example, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds.), pp. 243-56 (Alan R. Liss, Inc. (1985), Arnon et al, "Monoclonal Antibodies for Immunotargeting Of Drugs In Cancer, R., ed., R. Ern. Marcel Dekker, Inc., pp. 623-53 (1987), Hellstrom et al., "Antibodies For Drug Delivery", Monoclonal api., pp. 623-53, 1987. 506 (1985) Thorpe, "Antibody Carriers of Cytotoxic Agents In Cancer Therapy: A Reviews (16), 1985. Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy ", and, Immunol Rev...: Thorpe et al in (52 119-58 (1982))," the Preparation and Cytotoxic Properties of Antibody- See Toxin Conjugates".

[Bifunctional molecule]
PD-L1 is an immune checkpoint molecule and a tumor antigen. A bispecific antibody can be produced by combining an antibody or antigen-binding fragment specific for PD-L1 as a tumor antigen target molecule with a second antigen-binding fragment specific for immune cells. ..

In some embodiments, the immune cells are the group consisting of T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils and mast cells. Selected from. Molecules on immune cells that can be targeted include, for example, CD3, CD16, CD19, CD28 and CD64. Other examples are PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer cell immunoglobulin-like receptor (KIR), And CD47 is included. Specific examples of bispecificities include, but are not limited to, PD-L1/PD-1, PD-L1/LAG3, PD-L1/TIGIT, and PD-L1/CD47.

As an immune checkpoint inhibitor, an antibody or antigen-binding fragment specific for PD-L1 can be combined with a second antigen-binding fragment specific for a tumor antigen to produce a bispecific antibody. A "tumor antigen" is an antigenic substance produced in tumor cells, ie, it elicits an immune response in the host. Tumor antigens are useful in identifying tumor cells and are potential candidates for use in cancer therapy. The normal proteins in the body are not antigenic. However, certain proteins are produced or overexpressed during tumorigenesis and therefore appear "foreign" to the body. This can be a normal protein that is well isolated from the immune system, a protein that is usually produced in very small amounts, a protein that is usually produced only at certain stages of development, or a structure that is due to mutations. May include a modified protein.

Large amounts of tumor antigens are known in the art and new tumor antigens can be easily identified by screening. Non-limiting examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2. , GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin.

In some embodiments, the monovalent monofunctional has specificity for a protein overexpressed on tumor cells as compared to the corresponding non-tumor cells. As used herein, "corresponding non-tumor cell" refers to a non-tumor cell that is of the same cell type as the origin of the tumor cell. Note that such proteins do not necessarily differ from tumor antigens. Non-limiting examples include (a) carcinoembryonic antigen (CEA) overexpressed in most colon, rectal, breast, lung, pancreatic, gastrointestinal carcinomas, (b) breast, ovary, colon, lung. , Heregulin receptors (HER-2, neu or c-erbB-2) frequently overexpressed in prostate and cervical cancers, (c) chest, head and neck, non-small cell lung, and prostate Growth factor receptor (EGFR), which is highly expressed in various solid tumors, (d) asialoglycoprotein receptor, (e) transferrin receptor, (f) serpin enzyme complex receptor expressed in hepatocytes, (g) Of fibroblast growth factor receptor (FGFR) overexpressed in pancreatic ductal adenocarcinoma cells, (h) vascular endothelial growth factor receptor (VEGFR) for anti-angiogenic gene therapy, (i) non-mucin producing ovarian carcinoma Folate receptors selectively overexpressed in 90%, (j) cell surface glycocalyx, (k) carbohydrate receptors, (l) useful for gene transfer to respiratory epithelial cells, lungs such as cystic fibrosis It includes high molecular immunoglobulin receptors that are attractive for the treatment of disease. In this regard, non-limiting examples of bispecificities include PD-L1/EGFR, PD-L1/Her2, PD-L1/CD33, PD-L1/CD133, PD-L1/CEA, and PD- L1/VEGF is included.

Different forms of bispecific antibodies are also provided. In some embodiments, each of the anti-PD-L1 fragment and the second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

Bifunctional molecules that do not include only antibodies or antigen-binding fragments are also provided. Antibodies or antigen-binding fragments specific for PD-L1, such as those described herein, as tumor antigen targeting molecules can be combined with immune cytokines or ligands, optionally through a peptide linker. Linked immune cytokines or ligands include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL- 13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. Such bifunctional molecules can combine the immune checkpoint blocking effect with tumor site local immunomodulation.

[Polynucleotide encoding antibody and method for preparing antibody]
The disclosure also provides isolated polynucleotides or nucleic acid molecules (eg, SEQ ID NOs: 34-60, 112 and 114) encoding the antibodies, variants or derivatives of the disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen binding polypeptide, variants or derivatives thereof, either on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present disclosure include portions of the heavy and light chain variable regions of the antigen binding polypeptide, variants or derivatives thereof, either on the same polynucleotide molecule or on separate polynucleotide molecules. Can be coded.

Methods of making antibodies are known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen binding polypeptides of the present disclosure are fully human. Fully human antibodies can be made as described herein using techniques described in the art. For example, a fully human antibody against a specific antigen can be prepared by administering the antigen to a transgenic animal that produces such antibody in response to challenge but has been modified to lack an endogenous locus. .. Exemplary techniques that can be used to make such antibodies are described in US Pat. Nos. 6,150,584, 6,458,592, 6,420,140, which are incorporated by reference in their entirety. ing.

In certain embodiments, the prepared antibody does not elicit an adverse immune response in the animal to be treated, eg, human. In one embodiment, the antigen-binding polypeptides of the present disclosure, variants or derivatives thereof are modified to reduce immunogenicity by using art-recognized techniques. For example, the antibody can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies retain, or substantially retain, the antigen binding properties of the parent antibody, but are less immunogenic in humans, typically non-human antibodies such as mouse or primate antibodies. Derived from. This involves (a) transplanting the entire non-human variable domain into a human constant region to produce a chimeric antibody, (b) at least a portion of one or more of the non-human complementarity determining regions (CDRs) Grafting on human frameworks and constant regions, with or without framework residues, or (c) grafting entire non-human variable domains, but by replacing surface residues those in human-like regions. Can be accomplished by a variety of methods, including "cloaking". Such methods are described by Morrison et al. , Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al. , Adv. Immunol. 44:65-92 (1988); Verhoeyen et al. , Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994) and US Pat. Nos. 5,585,089, 5,693,761, 5,693,762 and 6,190,370, all of which are incorporated herein by reference. The entirety of which is incorporated herein by reference.

Deimmunization can also be used to reduce the immunogenicity of an antibody. As used herein, the phrase "deimmunization" includes modifying an antibody to modify a T cell epitope (see, eg, International Patent Application Publication Nos. WO/9852976 A1 and WO/0034317 A2). .. For example, the variable heavy and variable light chain sequences of the starting antibody were analyzed and the human T of each V region, showing the position of the epitope and other important residues within the sequence associated with the complementarity determining regions (CDRs). A cell epitope "map" is created. Individual T cell epitopes in the T cell epitope map are analyzed to identify alternative amino acid substitutions that have a low risk of altering the activity of the final antibody. A variety of alternative variable heavy chain and variable light chain sequences, including combinations of amino acid substitutions, have been designed that are later incorporated into various binding polypeptides. Typically between 12 and 24 variant antibodies are produced and tested for binding and/or function. The complete heavy and light chain genes contain modified variable regions and human constant regions are cloned into expression vectors, followed by introduction of the plasmid into cell lines to produce whole antibodies. The antibodies are then compared in appropriate biochemical and biological assays to identify optimal variants.

The binding specificity of the antigen binding polypeptides of the present disclosure can be determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme linked immunosorbent assay (ELISA).

Alternatively, the techniques described for the production of single-stranded units (US Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989)) may be applied to produce single-stranded units of the present disclosure. Single chain units are formed by linking the heavy and light chain fragments of the Fv region via amino acid bridges, resulting in a single chain fusion peptide. Techniques for assembling functional Fv fragments in E. coli can be used (Skerra et al., Science 242: 1038-1041 (1988)).

Examples of techniques that can be used to produce single chain Fv (scFv) and antibodies are described in US Pat. Nos. 4,946,778 and 5,258,498; Huston et al. , Methods in Enzymology 203:46-88 (1991); Shu et al. , Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al. , Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different parts of the antibody are derived from different animal species, such as an antibody having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region. Methods of producing chimeric antibodies are known in the art. For example, Morrison, Science 229: 1202 (1985); Oi et al., which is incorporated herein by reference in its entirety. , BioTechniques 4:214 (1986); Gillies et al. , J. Immunol. See Methods 125:191-202 (1989); U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397.

A humanized antibody is an antibody derived from a non-human species antibody that has a non-human species and framework regions and one or more complementarity determining regions (CDRs) of a human immunoglobulin molecule and binds to a desired antigen. It is a molecule. Framework residues in the human framework regions are often replaced with the corresponding CDR donor antibody residues to alter, preferably improve, antigen binding. These framework substitutions are identified by methods known in the art, for example, by modeling the interaction of CDRs with framework residues to identify framework residues important for antigen binding. , And sequence comparisons are used to identify unusual framework residues at specific positions. (See, eg, Queen et al., US Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which is incorporated herein by reference in its entirety). , CDR transplantation (EP 239,400; PCT publication WO 91/09967, US Pat. Nos. 5,225,539, 5,530,101; and 5,585,089), veneering or resurfacing. (EP 592, 106; EP 519, 596, Padlan, Molecular Immunology 28 (4/5): 489-498 (1991), Studnikka et al., Protein Engineering 7(6): 805-814 (1994), Ro. et al., Proc. Natl. Sci. USA 91:969-973 (1994)), including chain shuffling (US Pat. No. 5,565,332, incorporated herein by reference in its entirety). It can be humanized using various techniques known in the art.

Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. U.S. Pat. See also 33735 and WO 91/10741. Each of these is hereby incorporated by reference in its entirety.

Also, transgenic mice that are incapable of expressing functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes can be used to generate human antibodies. For example, the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, in addition to the human heavy and light chain genes, human variable regions, constant regions, diversity regions can be introduced into mouse embryonic stem cells. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region prevent the production of endogenous antibodies. Chimeric mice are made by growing modified embryonic stem cells and microinjecting them into blastocysts. Next, the chimeric mice are bred to produce homozygous progeny expressing the human antibody. For example, a selected antigen, such as all or part of the desired target polypeptide, is used to immunize transgenic mice in the usual fashion. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice rearrange during B cell differentiation, followed by class switching and somatic mutations. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed description of this technique for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, eg, PCT Publication WO 98, which is incorporated herein by reference in its entirety. /24893, WO 96/34096, WO 96/33735, U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,. See 661, 016, 5,545,806, 5,814,318 and 5,939,598. In addition, Abgenix, Inc. Companies such as (Fremont, Calif.) GenPharm (San Jose, Calif.) can engage in the provision of human antibodies to selected antigens using techniques similar to those described above.

Fully human antibodies that recognize selected epitopes can also be generated using the technique called "guided selection." In this approach, selected non-human monoclonal antibodies, such as mouse antibodies, are used to guide the selection of fully human antibodies that recognize the same epitope. (Jespers et al., Bio/Technology 72:899-903 (1988). See also US Pat. No. 5,565,332, which is incorporated by reference in its entirety.

In another embodiment, the DNA encoding the desired monoclonal antibody is labeled with an oligonucleotide probe capable of specifically binding to the genes encoding the heavy and light chains of the mouse antibody using conventional procedures. It can be easily isolated and sequenced (by use). The isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector, which in turn does not produce immunoglobulins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. , Into prokaryotic or eukaryotic host cells. More specifically, the isolated DNA, which may be synthesized as described herein, is disclosed in Newman et al., filed January 25, 1995, which is incorporated herein by reference. Can be used to clone constant and variable region sequences to produce antibodies as described in US Pat. Basically, this involves extraction of RNA from selected cells, conversion to cDNA, amplification by PCR using Ig-specific primers. Suitable primers for this purpose are also described in US Pat. No. 5,658,570. As described in more detail below, transformed cells expressing the desired antibody can be grown in relatively large quantities to provide a clinical and commercial supply of immunoglobulins.

In addition, by using routine recombinant DNA techniques, one or more of the CDRs of the antigen binding polypeptides of the present disclosure can be incorporated into framework regions, such as human, to humanize non-human antibodies. It can be inserted in the framework region. The framework regions can be naturally occurring or consensus framework regions, preferably the human framework regions (a list of human framework regions can be found, for example, in Chothia et al., J. Mol. Biol. 278:457-479 (1998)). Preferably, the polynucleotide produced by the combination of the framework regions and the CDRs encodes an antibody that specifically binds at least one epitope of the desired polypeptide, such as LIGHT. Preferably, one or more amino acid substitutions can be made within the framework regions, preferably the amino acid substitutions improve the binding of the antibody to the antigen. In addition, such methods make amino acid substitutions or deletions of one or more variable region cysteine residues involved in intrachain disulfide bonds to produce antibody molecules lacking one or more intrachain disulfide bonds. Can be used for. Other changes to the polynucleotide are encompassed by this disclosure and within the skill of the art.

In addition, techniques developed to generate "chimeric antibodies" by splicing the gene for a mouse antibody molecule with the appropriate antigen specificity along with the gene for a human antibody molecule of suitable biological activity. (Morrison et al., Proc. Natl. Acad. Sci. USA: 851-855 (1984), Neuberger et al., Nature 372:604-608 (1984), Takeda et al., 3: 45). 454 (1985)). As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region.

Yet another very efficient means for producing recombinant antibodies is disclosed in Newman, Biotechnology 10: 1454-1460 (1992). Specifically, this technique results in primatized antibodies that contain monkey variable domains and human constant sequences. This reference is incorporated herein by reference in its entirety. In addition, this technique is also described in commonly assigned US Pat. Nos. 5,658,570, 5,693,780 and 5,756,096, each incorporated herein by reference. It

Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to those of ordinary skill in the art. Such techniques are described in various laboratory manuals and primary publications. In this regard, suitable techniques for use in the disclosure set forth below are described in Current Protocols in Immunology, Coligan et al. , Eds. , Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference in its entirety, including the appendices.

In addition, it is known to those skilled in the art to introduce mutations into the nucleotide sequences encoding the antibodies of the present disclosure, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis that cause amino acid substitutions. Standard techniques can be used. Preferably, the variant (including derivative) is a variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, variable light chain region, CDR-L1, CDR-L2, or CDR-L3 serving as a reference. To less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions Substitutions, <4 amino acid substitutions, <3 amino acid substitutions, or <2 amino acid substitutions are encoded. Alternatively, the mutations can be introduced randomly along all or part of the coding sequence, such as saturation mutagenesis, and the resulting variants screened for biological activity. It is possible to identify a mutant that retains.

[Cancer treatment]
As described herein, the antibodies, variants or derivatives of this disclosure can be used in certain therapeutic and diagnostic methods.

The disclosure further relates to antibody-based therapies that involve administering the disclosed antibodies to patients, such as animals, mammals and humans, to treat one or more of the disorders or conditions described herein. To do. Therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure, including the variants described herein and derivatives of the variants, and the antibodies of the present disclosure (herein. Nucleic acids or polynucleotides, including variants and derivatives of variants described in.

The antibodies of this disclosure can also be used to treat or inhibit cancer. PD-L1 can be overexpressed in tumor cells. Tumor-derived PD-L1 can bind to PD-1 on immune cells, thereby limiting anti-tumor T cell immunity. The results using small molecule inhibitors or monoclonal antibodies targeting PD-L1 in a mouse tumor model indicate that PD-L1 targeted therapeutics are important alternatives and realities for effective control of tumor growth. It shows that it is a technical method. As shown in the experimental examples, anti-PD-L1 antibodies activate a mechanism of adaptive immune response that can lead to improved survival in cancer patients.

Thus, in some embodiments, methods are provided for treating cancer in a patient in need thereof. In one embodiment, the method involves administering to a patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the cancer cells (eg, stromal cells) in the patient expresses, overexpresses, or is induced to express PD-L1. Induction of PD-L1 expression can be performed, for example, by administration of a tumor vaccine or radiotherapy.

Tumors expressing PD-L1 protein include bladder cancer, non-small cell lung cancer, renal cancer, breast cancer, urethral cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma, Merkel cell carcinoma, gastrointestinal cancer, gastric cancer, Includes esophageal cancer, ovarian cancer, renal cancer, small cell lung cancer. Accordingly, the antibodies of the present disclosure can be used to treat any one or more such cancers.

Cellular therapies such as chimeric antigen receptor (CAR) T cell therapies are also provided in the present disclosure. Suitable cells can be used, which are contacted (or, alternatively, engineered to express an anti-PD-L1 antibody of the present disclosure). Following such contact or manipulation, cells can be introduced into a cancer patient in need of treatment. A cancer patient can have any type of cancer as disclosed herein. The cell (eg, T cell) can be, for example, but not limited to, a tumor infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or a combination thereof.

In some embodiments, the cells have been isolated from the cancer patient itself. In some embodiments, cells are provided by a donor or from a cell bank. When isolating cells from cancer patients, unwanted immune reactions can be minimized.

Additional diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed, and/or prognosed with the antibodies or variants of the present disclosure, or derivatives thereof, include, but are not limited to: , Leukemia (acute leukemia, (eg, acute lymphocytic leukemia, acute myelogenous leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia)), chronic leukemia (eg, (Including chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (eg Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulin blood Disease, heavy chain disease, and solid tumors, including but not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelium, Synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, malignant melanoma, prostate cancer, ovarian cancer, prostate cancer, flat Epithelial cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, villi Cancer, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma Tumors, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, malignant melanomas, sarcomas such as neuroblastomas, retinoblastomas and carcinomas) The associated disease progression and/or metastasis is included.

[Combination therapy]
In a further embodiment, the compositions of the present disclosure are administered in combination with an antitumor agent, antiviral agent, antibacterial or antibiotic agent, or antifungal agent. Any of these formulations known in the art can be administered in the compositions of the present disclosure.

In another embodiment, the compositions of this disclosure are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the compositions of the present disclosure include, but are not limited to, antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, dactinomycin), anti-estrogens (eg, tamoxifen), antimetabolites. (Eg, fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, 6-thioguanine), cytotoxic agent (eg, carmustine, BCNU, lomustine, CCNU, cytosine arabi) Nocid, cyclophosphamide estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, vincristine sulfate), hormones (eg, medroxyprogesterone, estramustine phosphate phosphate, ethinyl estradiol, estradiol, megestrol acetate). , Methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, test lactone), nitrogen mustard derivatives (eg melphalan, chlorambucil, mechlorethamine (nitrogen mustard), thiotepa), steroids and combinations (eg phosphate) Betamethasone sodium) and others (for example, dacarbazine, asparaginase, mitotan, vincristine sulfate, vinblastine sulfate, etoposide) and the like.

In a further embodiment, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12. , IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

In a further embodiment, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic modalities such as, for example, radiation therapy.

Combination therapy comprising using one or more of the anti-PD-L1 antibodies of the present disclosure with a second anti-cancer (chemotherapy) agent is also provided. Chemotherapeutic agents, for example, are antimetabolites/anticancer agents such as the pyrimidine analogues floxuridine, capecitabine, cytarabine; purine analogues, folate antagonists and related inhibitors; vinca alkaloids (vinblastine, vincristine). ) And antiproliferative/anti-antimicrobial agents such as taxanes (paclitaxel, docetaxel), vinblastine, nocodazole, epothilone, vinorelbine (NAVELBINE®), epipodophyllotoxin (etoposide, teniposide). Mitotic agents; actinomycin, amsacrine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide (CYTOXAN (registered trademark)), dactinomycin, daunorubicin, doxorubicin, epirubicin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitokitin. DNA damaging agents such as santorone, nitrosourea, procarbazine, taxol, taxotere, teniposide, etoposide, triethylenethiophosphoramide; dactinomycin, daunorubicin, doxorubicin, idarubicin, anthracycline, mitoxantrone, bleomycin, plicamycin (mitra. (Mycin), mitomycin, and other antibiotics; enzymes that metabolize L-asparagine systemically and remove cells that do not have the ability to synthesize their own asparagine; antiplatelets; nitrogen mustard cyclophosphamide And anti-proliferative/anti-mitotic alkylating agents such as analogs (melphalan, chlorambucil, hexamethylmelamine, thiotepa), alkylnitrosourea (carmustine) and analogs, streptozocin, triazene (dacarbazine); folate analogs ( Antiproliferative/antimitotic antimetabolites such as methotrexate); platinum coordination complexes (cisplatin, oxaliplatin, carboplatin), procarbazine, hydroxyurea, mitotan, aminoglutethimide; hormones, hormone analogues (estrogens, tamoxifen, Goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole and anastrozole); anticoagulants such as heparin, synthetic heparin salts, and other inhibitors of thrombin; tissue plasminogen activator, strept Kinase, urokinase, aspirin, dipyridamole, ticlopidine , Fibrinolytic agents such as clopidogrel; anti-migratory agents; anti-secretory agents (brefeldin); immunosuppressive agents tacrolimus, sirolimus, azathioprine, mycophenolic acid; compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth) Factor inhibitors and fibroblast growth factor inhibitors); angiotensin receptor blockers, nitric oxide donors; antisense oligonucleotides; antibodies such as trastuzumab and rituximab; cell cycle inhibitors such as tretinoin and differentiation inducers; inhibitors; Topoisomerase inhibitors (doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone Factor signaling kinase inhibitors; dysfunction-inducing factors; cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin, caspase activator; and chromatin.

Further, examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, piposulfan; benzodopa, carbocon, metredopa, uredopa and the like. Aziridine; altrelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, emilermine including trimethylolmelamine and memiramelamin; acetogenin (especially bratacin and bratacinone); camptothecin (including synthetic analog topotecan); bryostatin; Kallistatin; CC-1065 (including its adzeresin, calzeresin, and bizeresin synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (synthetic analogs KW-2189 and CBI-TMI ); eloiterobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornafazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbikin, phenesterine. , Nitrogen mustards such as predonimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; enediyne antibiotics (eg calicheamicin, especially calicheamicin γa2 and calicheamicin Φ1). , Dinemycin including dinemycin A, bisphosphonates such as clodronate, esperamycin, neocarzinostatin chromophore and related chromoprotein enediin antibiotics chromophore, acracinomycin, actinomycin, ausramycin, azaserine, bleomycin, kakuti Nomycin, carabicin, carminomycin, cardinophylline, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- (Including doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C and other mitomycin, mycoph Antibiotics such as enolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, queramycin, rodrubicin, streptonigrins, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; methotrexate and 5-fluorouracil (5-FU) Antimetabolites such as; folic acid analogs such as demopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxy. Pyrimidine analogues such as uridine, doxyfluridine, enocitabine, and floxuridine; androgens such as carsterone, dromostananolone propionate, epithiostanol, mepithiostane, and test lactone; antiadrenal agents such as aminoglutethimide, mitotan, and trilostane; Folic acid supplements such as frolic acid); trichothecenes (particularly T-2 toxin, veracrine A, loridin A, anguidine); taxoids such as paclitaxel (Taxol®) and docetaxel (Taxotere®); cisplatin and carboplatin Platinum analogs such as; asegraton; aldophosphamide glycoside; aminolevulinic acid; enyluracil; amsacrine; hestrabucil; bisanthrene; edatraxate; defofamine (defofamine); demecorcin; diazicon; elformin; elliptinium acetate; epothilone nitrate, etogluside; Gallium; hydroxyurea; lentinan; leucovorin; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidammol; nitracrine; pentostatin; phenamet; pirarubicin; rosoxantrone; fluoropyrimidine; folinic acid; podophyllic acid; 2- Ethylhydrazide; Procarbazine; Polysaccharide-K (PSK); Razoxane; Rhizoxine; Schizophyllan; Spirogermanium; Tenuazoic acid; Triadicon; 2,2',2''-Triquorotriemylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside (“Ar a-C"); cyclophosphamide; thiopeta; chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; Vincristine; vinorelbine (NAVELBINE®); Novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; Capecitabine; FOLFIRI (fluorouracil, leucovorin, irinotecan); and any of the above pharmaceutically acceptable salts, acids or derivatives, and the like.

In addition, the definition of "chemotherapeutic agent" includes an anti-estrogen agent, a selective estrogen receptor modulator (SERM), an inhibitor of the enzyme aromatase, an anti-androgen agent, and a hormonal action on a tumor. Antihormonal agents such as any of the above pharmaceutically acceptable salts, acids or derivatives that function in

Examples of anti-estrogens and SERMs include, for example, tamoxifen (including Nolvadex®), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxyphene, cheoxyphene, LY117018, onapristone and toremifene (fairstone (Registered trademark)) is included.

Inhibitors of the enzyme aromatase regulate estrogen production in the adrenal gland. Examples are 4(5)-imidazole, aminoglutethimide, megestrol acetate (MEGACE®), exemestane, formestane, fadrozole, borozole (RIVISOR®), letrozole (FEMARA®). )), and anastrozole (ARIMIDEX®).

Examples of anti-androgens include flutamide, nilutamide, bicalutamide, leuprolide, goserelin.

Examples of chemotherapeutic agents include, but are not limited to, retinoid acid and its derivatives 2-methoxyestradiol, angiostatin®, endostatin®, suramin, squalamine, metalloproteinase-1. Tissue inhibitor, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, cartilage-derived inhibitor, paclitaxel (nab-paclitaxel), platelet No. 4 Factor, protamine sulfate (curpain), sulfated chitin derivative (prepared from snow crab shell), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, proline analogue (L-azetidine-2-carboxylic acid (LACA) )) containing modulators of substrate metabolism, cis-hydroxyproline, d,L-3,4-dehydroproline, thiaproline, α,α'-dipyridyl, β-aminopropionitrile fumarate, 4-propyl-5-( 4-pyridinyl)-2(3h)-oxazolone, methotrexate, mitoxantrone, heparin, interferon, 2 macroglobulin serum, chicken metalloproteinase-3 inhibitor (ChIMP-3), chymostatin, β-cyclodextrin tetradeca sulfate Salt, eponomycin, fumagillin, sodium gold thiomalate, d-penicillamine, β-1-anticollagenase serum, α-2-antiplasmin, bisanthrene, lobenzarit disodium, n-2-carboxyphenyl-4-chloroanthranilate disodium Alternatively (“CCA”), anti-angiogenic agents including metalloproteinase inhibitors such as thalidomide, anti-angiogenic steroids, carboxaminoimidazole, BB-94. Other anti-angiogenic agents include antibodies, preferably monoclonal antibodies against these angiogenic growth factors (β-FGF, α-FGF, FGF-5, VEGF isoforms, VEGF-C, HGF/SF, Ang- 1/Ang-2) is included.

Also, examples of chemotherapeutic agents include, but are not limited to, compounds such as β-aminopropionitrile (BAPN), and diseases and conditions associated with abnormal deposition of collagen, which are incorporated herein by reference. US Pat. No. 4,965,288 (Palfreyman, et al.) relating to inhibitors of lysyl oxidase and their use in the treatment of LOX and inhibiting LOX for the treatment of various pathological fibrotic conditions Included are anti-fibrotic agents that include compounds disclosed in US Pat. No. 4,997,854 (Kagan et al.), which pertains to compounds. Further, exemplary inhibitors relate to compounds such as 2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine, incorporated herein by reference, US Pat. No. 4,943,593 (Palfreyman et al), US Pat. No. 5,021,456 (Palfreyman et al.), 5,059,714 (Palfreyman et al.) relating to 2-(1-naphthyloxymethyl)-3-fluoroallylamine. .), 5,120,764 (McCarthy et al.), 5,182,297 (Palfreyman et al.), 5,252,608 (Palfreyman et al.), US Patent Publication 2004. /0248871 (Farjanel et al.).

Exemplary anti-fibrotic agents also produce a primary amine that reacts with the carbonyl group of the active site of lysyl oxidase, more specifically, after binding with a carbonyl, produces a resonance-stabilized product. , Emylenemmamine, hydrazine, phenylhydrazine and their derivatives; semicarbazide and urea derivatives; amino nitriles such as BAPN or 2-nitroethylamine; 2-bromo-ethylamine, 2-chloroethylamine, 2-trifluoroethylamine, Unsaturated or saturated haloamines such as 3-bromopropylamine and p-halobenzylamine; primary amines such as selenohomocysteine lactone are included.

Other anti-fibrotic agents are copper chelators that either penetrate or do not penetrate cells. Exemplary compounds include indirect inhibitors that block aldehyde derivatives that result from the oxidative deamination of lysyl and hydroxylysyl residues by lysyl oxidase. Examples are thiolamines, especially d-penicillamine, and 2-amino-5-mercapto-5-methylhexanoic acid, D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio). Butyric acid, p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butyric acid, sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane sulfide Included are analogs thereof such as 2-acetamidoethyl-2-acetamidoethanethiol sulfanate, sodium-4-mercaptobutane sulfinate trihydrate and the like.

Examples of chemotherapeutic agents also include immunotherapeutic agents including, but not limited to, therapeutic antibodies suitable for treating the patient. Some examples of therapeutic antibodies, Shimutsuzumabu, Abagobomabu, Adekatsumumabu, Afutsuzumabu, alemtuzumab, Arutsumomabu, Amatsukishimabu, Anatsumomabu, Arushitsumomabu, Babitsukishimabu, Bekutomomabu, bevacizumab, Bibatsuzumabu, Burinatsumomabu, Brent screeching blanking, Kantsuzumabu, Katsumakisomabu, cetuximab, Shitatsuzumabu , Shikutsumumabu, Kuribatsuzumabu, Konatsumumabu, Daratsumabu, Dorojitsumabu, Dorigotsumabu, Deyushigitsumabu, Detsumomabu, Dasetsuzumabu, Darotsuzumabu, Ekuromekishimabu, Erotsuzumabu, Enshitsukishimabu, Erutsumakisomabu, Etarashizumabu, Faretsuzumabu, Fikuratsuzumabu, Figitsumumabu, Furanbotsumabu, Futsukishimabu, Ganitsumabu, gemtuzumab, Girentsukishimabu, Gurenbatsumumabu , ibritumomab, Igobomabu, Imatsuzumabu, Indatsukishimabu, Inotsuzumabu, Intetsumumabu, ipilimumab, Iratsumabu, labetuzumab, lexatumumab, lintuzumab, Rorubotsuzumabu, Rukatsumumabu, mapatumumab, matuzumab, milatuzumab, Minretsumamabu, Mitsumomabu, Mokisetsumomabu, Narunatsumabu, Naputsumomabu, Nechitsumumabu, nimotuzumab, Nofetsumomabu, Okaratsuzumabu , Ofuatsumumabu, Oraratsumabu, Onatsuzumabu, Oporutsuzumabu, oregovomab, panitumumab, Pasatsuzumabu, Patoritsumabu, pemtumomab, pertuzumab, Pintsumomabu, Puritsumumabu, Rakotsumomabu, Radoretsumabu, Rirotsumumabu, rituximab, Robatsumumabu, Satsumomabu, sibrotuzumab, Shirutsukishimabu, Soritomabu, Takatsuzumabu, Tapuritsumomabu, Tenatsumomabu, Tepurotsumumabu , Tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ubrituximab, veltuzumab, borcetuzumab, botsumumab, saltumumab, CC49, 3F8. Rituximab can be used to treat indolent B-cell cancers including marginal zone lymphoma, WM, CLL, and small lymphocytic lymphoma. The combination of rituximab and chemotherapeutic agents is particularly effective.

Exemplary therapeutic antibodies can be further labeled with, or combined with, radioisotope particles such as indium-111, yttrium-90, or iodine-131.

In one embodiment, the additional therapeutic agent is a nitrogen mustard alkylating agent. Non-limiting examples of nitrogen mustard alkylating agents include chlorambucil.

In one embodiment, the compounds and compositions described herein can be used with, or combined with, one or more additional therapeutic agents. One or more therapeutic agents include, but are not limited to, inhibitors of Abl, activated CDC kinase (ACK), adenosine A2B receptor (A2B), apoptosis signal-regulating kinase (ASK), auroa kinase, Breton. Tyrosine kinase (BTK), BET-bromo domain (BRD) such as BRD4, c-Kit, c-Met, CDK activating kinase (CAK), calmodulin-dependent protein kinase (CaMK), cyclin-dependent kinase (CDK), Casein kinase (CK), discoidin domain receptor (DDR), epidermal growth factor receptor (EGFR), focal adhesion kinase (FAK), Flt-3, FYN, glycogen synthase kinase (GSK), HCK, histone Acetylase (HDAC), IKK such as IKKβε, isocitrate dehydrogenase (IDH) such as IDH1, Janus kinase (JAK), KDR, lymphocyte-specific protein tyrosine kinase (LCK), lysyl oxidase protein, lysyl oxidase-like protein (LOXL) ), LYN, matrix metalloprotease (MMP), MEK, mitogen-activated protein kinase (MAPK), NEK9, NPM-ALK, p38 kinase, platelet-derived growth factor (PDGF), phosphorylase kinase (PK), polo-like kinase (PLK) ), phosphatidylinositol 3-kinase (PI3K), protein kinases such as protein kinases A, B and/or C (PK), PYK, spleen tyrosine kinase (SYK), serine/threonine kinase TPL2, serine/threonine kinase STK, signal Transduction and transcriptional activator (STAT), SRC, serine/threonine protein kinases (TBK) such as TBK1, TIE, tyrosine kinase (TK), vascular endothelial growth factor receptor (VEGFR), YES, or any combination thereof Is included.

ASK inhibitors include ASK1 inhibitors. Examples of ASK1 inhibitors include, but are not limited to, those described in WO 2011/008709 (Gilead Sciences) and WO 2013/112741 (Gilead Sciences).

Examples of BTK inhibitors include, but are not limited to, ibrutinib, HM71224, ONO-4059 and CC-292.

DDR inhibitors include inhibitors of DDR1 and/or DDR2. Examples of DDR inhibitors include, but are not limited to, WO 2014/047624 (Gilead Sciences), US 2009/0142345 (Takeda Pharmaceutical), US 2011/0287011 (Oncomedpharmaceutical) 02, WO 2013/0142745, and WO 2013/02. Included are those disclosed in WO 2013/034933 (Imperial Innovations).

Examples of HDAC inhibitors include, but are not limited to, plasinostat and panobinostat.

The JAK inhibitor inhibits JAK1, JAK2, and/or JAK3. Examples of JAK inhibitors include, but are not limited to, filgotinib, ruxolitinib, fedratinib, tofacitinib, baricitinib, lestaurtinib, paclitinib, XL019, AZD1480, INCB039110, LY2784544, BMS8911543 and NS01.

LOXL inhibitors include inhibitors of LOXL1, LOXL2, LOXL3, LOXL4, and/or LOXL5. Examples of LOXL inhibitors include, but are not limited to, the antibodies described in WO 2009/017833 (Arresto Biosciences).

Examples of LOXL2 inhibitors include, but are not limited to, the antibodies described in WO 2009/017833 (Arresto Biosciences), WO 2009/035791 (Arresto Biosciences) and WO 20111/097513 (Gilead Biologics).

MMP inhibitors include inhibitors of MMP1-10. Examples of MMP9 inhibitors include, but are not limited to, marimasterat (BB-2516), cipemasterat (Ro 32-3555), and those described in WO 2012/027721 (Gilead Biologies).

PI3K inhibitors include inhibitors of PI3Kγ, PI3Kδ, PI3Kβ, PI3Kα, and/or pan-PI3K. Examples of PI3K inhibitors include, but are not limited to, wortmannin, BKM120, CH51332799, XL756 and GDC-0980.

Examples of PI3Kγ inhibitors include, but are not limited to, ZSTK474, AS252424, LY294002 and TG100115.

Examples of PI3Kδ inhibitors include, but are not limited to, PI3K II, TGR-1202, AMG-319, GSK2269557, X-339, X-414, RP5090, KAR4141, XL499, OXY111A, IPI-145, IPI-443. , And WO 2005/113556 (ICOS), WO 2013/052699 (Gilead Calistoga), WO 2013/116562 (Gilead Calistoga), WO 2014/100765 (Gilead Calistoga), WO 2014/100767 (Gilead Calito), and Compounds described in 2012409 (Gilead Sciences) are included.

Examples of PI3Kβ inhibitors include, but are not limited to, GSK2636771, BAY 10824391 and TGX221.

Examples of PI3Kα inhibitors include, but are not limited to, Buparricib, BAY 80-6946, BYL719, PX-866, RG7604, MLN1117, WX-037, AEZA-129 and PA799.

Examples of pan-PI3K inhibitors include, but are not limited to, LY294002, BEZ235, XL147 (SAR245408) and GDC-0941.

Examples of SYK inhibitors include, but are not limited to, tamatinib (R406), fostamatinib (R788), PRT062607, BAY-61-3606, NVP-QAB 205 AA, R112, R343, and U.S. Pat. No. 8,450. , 321 (Gilead Connecticut).

TKIs can target the epidermal growth factor receptor (EGFR) and the receptors for fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Examples of TKIs that target the EGFR include, but are not limited to, gefitinib and erlotinib. Sunitinib is a non-limiting example of a TKI that targets the receptors for FGF, PDGF and VEGF.

The anti-PD-L1 antibodies of the present disclosure can be used with immune checkpoint inhibitors in some embodiments. Immune checkpoints are molecules in the immune system that either increase the signal (costimulatory molecule) or decrease the signal (cosuppressor molecule). Many cancers protect themselves from the immune system by inhibiting T cell signaling through agonists for co-suppressor molecules or antagonists for costimulatory molecules. Immune checkpoint agonists or antagonists can help stop such protective mechanisms by the cell. Immune checkpoint agonists or antagonists include PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX -40/OX40L, CD40/CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), any of the checkpoint molecules One or more of these may be targeted.

Programmed T cell death 1 (PD-1) is a transmembrane protein found on the surface of T cells that inhibits T cell activity when bound to programmed T cell death ligand 1 (PD-L1) or tumor cells, Alternatively, it results in a decrease in T cell mediated cytotoxicity. Therefore, PD-1 and PD-L1 are immune down-regulators or "off-switches" of immune checkpoints. Examples of PD-1 inhibitors include, but are not limited to, nivolumab, (Opdivo) (BMS-936558), pembrolizumab (Keytruda), pidilizumab, AMP-224, MEDI0680 (AMP-514), PDR001, MPDL3280A, MEDI47. , BMS-936559 and MSB0010718C.

CTLA-4 is a protein receptor that downregulates the immune system. Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy®) (also known as BMS-734016, MDX-010, MDX-101), and tremelimumab (formerly ticilimumab, CP- 675,206) are included.

Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint receptor on the cell surface that functions to suppress immune responses by acting on regulatory T cells and directly on CD8+ T cells. is there. LAG-3 inhibitors include, but are not limited to, LAG525 and BMS-986016.

CD28 is constitutively expressed on almost all human CD4+ T cells and about half of all CD8 T cells. CD28 promotes T cell proliferation. A non-limiting example of a CD28 inhibitor includes TGN1412.

CD122 increases the proliferation of CD8+ effector T cells. Non-limiting examples include NKTR-214.

4-1BB (also known as CD137) is involved in T cell proliferation. CD137-mediated signaling is also known to protect T cells, especially CD8+ T cells, from activation-induced cell death. PF-05082566, urelumab (BMS-663513) and lipocalin are examples of CD137 inhibitors.

For any of the above combination therapies, the anti-PD-L1 antibody can be administered concurrently or separately with the other anti-cancer agent. When administered separately, the anti-PD-L1 antibody can be administered before or after the other anti-cancer agent.

[Treatment of infection]
As shown in the experimental examples, the antibodies of the present disclosure can activate immune responses that can be useful in treating infections.

Infection is the reaction of host tissue by pathogens to enter the body's tissues of organisms, their growth, and to these pathogens and the toxins they produce. Infection can occur with viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks, mites, fleas, lice, fungi such as ringworm, and other major parasites such as tapeworms and other helminths. It can be caused by an infectious agent such as an insect. In one aspect, the infectious agent is a bacterium such as a Gram-negative bacterium. In one aspect, infectious agents are viruses such as DNA viruses, RNA viruses, and reverse transcription viruses. Non-limiting examples of viruses include adenovirus, coxsackie virus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalo. Virus, human herpes virus type 8, HIV, influenza virus, measles virus, mumps virus, human papilloma virus, parainfluenza virus, poliovirus, rabies virus, RS virus, rubella virus, varicella-zoster virus.

The antibodies of the present disclosure can also be used to treat infections caused by or kill microbes by targeting the microbes and immune cells to effect elimination of the microbes. In one aspect, microorganisms include viruses, including RNA and DNA viruses, Gram-positive bacteria, Gram-negative bacteria, protozoa or fungi. Non-limiting examples of infectious diseases and related microorganisms are provided in Table 4 below.
[Table 4 Infectious Diseases and Related Sources of Microbial Infection]

The specific dose and treatment regimen for a particular patient are specific antibodies, the variant or derivative used, the patient's age, weight, general health, sex, diet, time of administration, excretion rate, concomitant medications. , Will depend on various factors, including the severity of the particular disease being treated. Judgment of such factors by medical personnel is within the ordinary competence in the field. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles known in the art.

Methods of administering the antibody or variant include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal lining, such as the oral mucosa, rectal and intestinal mucosa. , Can be administered with other biologically active agents. Accordingly, pharmaceutical compositions comprising the disclosed antigen-binding polypeptides may be administered orally, rectally, parenterally, intravesically, intravaginally, intraperitoneally, topically (as a powder, ointment, injectable or transdermal patch), orally. Alternatively, it can be administered as a buccal or nasal spray.

The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intraarticular injection and infusion.

Administration can be systemic or local. In addition, it may be desirable to introduce the antibodies of the present disclosure into the central nervous system by suitable routes including intraventricular and intrathecal injection. Intraventricular injection can be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as the Omaya reservoir. Pulmonary administration can also be employed, eg, by use of an inhaler or nebulizer, and formulation of an aerosol.

It may be desirable to administer the antibody polypeptides or compositions of the present disclosure locally to the area in need of treatment. This can be accomplished, for example, but not limited to, by local injection during surgery, topical application (eg, in combination with post-surgical wound dressing), injection, by catheter, by suppository, or by implant, as described above. Implants are porous, non-porous, or gel-like materials that include membranes or fibers such as sialastic membranes. Preferably, when administering proteins of the present disclosure, including antibodies, care must be taken to use materials that do not absorb the protein.

In another embodiment, the antibody or composition is capable of being transported in vesicles, particularly liposomes (Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease, Los Angeles, and Los Angeles, USA). -Berstein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berstein, ibid., pp. 317-327; see generaly ibid.

In yet another embodiment, the antigen binding polypeptide or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. 1989. J. Med. 321:574). In another embodiment, a polymeric material may be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Rue de Lau, et al. (1974); Controlled bureau de Bruges). , Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. , 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, the controlled release system can be placed in the vicinity of the treated subject, ie, the brain, thus requiring only a fraction of the case of systemic dose administration (eg Goodson, in Medical). Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are described in the review of Langer (1990, Science 249:1527-1533).

In certain embodiments, where the composition of the present disclosure comprises a nucleic acid or a polynucleotide encoding a protein, the nucleic acid is constructed such that it is part of a suitable nucleic acid expression vector such that it is intracellular. Administration (eg, by use of a retroviral vector (see US Pat. No. 4,980,286)), by direct injection, or by use of microparticle bombardment (eg, gene gun, Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfecting agents, or by binding to homeobox-like peptides known to enter the nucleus and administration (eg, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), nucleic acids can be administered in vivo to promote expression of the encoded protein. Alternatively, the nucleic acid can be introduced intracellularly and integrated into the host cell DNA for expression by homologous recombination.

The amount of antibodies of the present disclosure that will be effective in treating, suppressing and preventing an inflammatory, immune or malignant disease, disorder or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose utilized in a formulation will depend on the route of administration and the severity of the disease, disorder or condition and should be decided according to the judgment of the practitioner and the circumstances of the individual patient. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

As a general proposition, the dose of the antigen binding polypeptide of the present disclosure administered to a patient is typically between 0.1 mg/kg and 100 mg/kg of the patient's body weight. To 0.1 mg/kg to 20 mg/kg, or to the patient's body weight between 1 mg/kg and 10 mg/kg. In general, human antibodies have a longer half-life within the human body than antibodies from other species. This is due to the immune response to the foreign polypeptide. Therefore, lower dosages of human antibodies and less frequent administration is often possible. In addition, the dosage and frequency of administration of the antibodies of the present disclosure may be reduced by improving absorption (eg, into the brain) and tissue permeability of the antibody, eg, by modification such as lipidation.

Methods for treating infectious or malignant diseases, conditions or disorders, including administration of the antibodies, variants or derivatives of the present disclosure, are typically tested in vitro and then used in humans. Prior to testing for the desired therapeutic or prophylactic activity in vivo in an acceptable animal model. Suitable animal models, including transgenic animals, are known to those of skill in the art. For example, in vitro assays demonstrating the therapeutic utility of antigen-binding polypeptides described herein include the action of the antigen-binding polypeptide on cell lines or tissue samples of patients. The effect of an antigen binding polypeptide on a cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art, such as the assays disclosed elsewhere herein. In accordance with the present disclosure, in vitro assays that can be used to determine if administration of a particular antigen binding polypeptide are indicated include in vitro cell culture assays, in which patient tissue samples are cultured. Are grown in and exposed to the compound, or otherwise administered, and the effect of the compound on the tissue sample is observed.

See, eg, encapsulation within liposomes, microparticles, microcapsules, recombinant cells capable of expressing compounds, receptor-mediated endocytosis (see, eg, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432). ), construction of nucleic acids as part of a retrovirus or other vector, such as administration of an antibody of the present disclosure or a polynucleotide encoding the antibody of the present disclosure. Can be used for

[Diagnostic method]
PD-L1 overexpression was observed in certain tumor samples, and patients with PD-L1 overexpressing cells may respond to treatment with anti-PD-L1 antibodies of the present disclosure. Accordingly, the antibodies of the present disclosure can also be used for diagnostic and prognostic purposes.

The sample, preferably containing cells, can be obtained from a patient, which can be a cancer patient or a patient for whom a diagnosis is desired. The cells can be cells of tumor tissue or tumor mass, blood samples, urine samples, or any sample from a patient. After any pretreatment of the sample, the sample can be incubated with the antibodies of the present disclosure under conditions that allow the antibody to interact with PD-L1 proteins potentially present in the sample. The presence of PD-L1 protein in the sample can be detected using methods such as ELISA, which utilize anti-PD-L1 antibodies.

The presence (optionally, amount or concentration) of PD-L1 protein in the sample is indicative of the patient's suitability for treatment with the antibody or the patient has responded (or did not respond) to cancer treatment. ) Can be used for cancer diagnosis. For prognostic methods, detection can be performed once, twice, or more at specific stages after the start of cancer treatment to indicate the progress of the treatment.

[composition]
The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (eg, immune checkpoint inhibitor).

In certain embodiments, the phrase "pharmaceutically acceptable" is approved by a federal or state governmental regulatory agency for use in animals, and more specifically in humans, or at the US Pharmacy. Means that it is listed in the Society or other generally recognized pharmacopoeia. Furthermore, a "pharmaceutically acceptable carrier" is generally a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any kind. ..

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. The pharmaceutical carrier may be a sterile liquid such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. In addition, saline and aqueous solutions of dextrose and glycerol can be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk powder, glycerol, Includes propylene, glycol, water, ethanol and the like. The composition may, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citric acid, or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and osmolality adjusting agents such as sodium chloride or dextrose are also envisioned. These compositions may take the form of solutions, suspensions, emulsions, tablets, tablets, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium, stearate, sodium saccharin, cellulose, magnesium carbonate. Examples of suitable pharmaceutical carriers are described in E.I. W. It is described in Remington's Pharmaceutical Sciences by Martin. Such compositions will contain a therapeutically effective amount of the antigen binding polypeptide, preferably in purified form, in combination with a suitable amount of carrier so as to provide the form for proper administration to the patient. Will be included. The formulation should suit the mode of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile aqueous isotonic buffer. The composition, if needed, can also include a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the site of the injection. Generally, the components are supplied separately or mixed together in unit dosage form, for example, as a lyophilized powder or anhydrous concentrate, in a sealed container such as an ampoule or sachet indicating the quantity of active agent. Either is done. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochlorides, phosphates, acetates, oxalates, tartrates, sodium, potassium, ammonium, calcium, Those formed with cations, such as those derived from ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. are included.

[Example]
[Example 1: Generation of human monoclonal antibody against human PD-L1]
Anti-human PD-L1 mouse monoclonal antibody is produced using hybridoma technology.

Antigen: CHOK1 cell line highly expressing human PDL1-Fc protein and human PD-L1 (PDL1-CHOK1 cell line).

Immunization: To generate mouse monoclonal antibodies against human PD-L1, 6-8 week old female BALB/c mice were first immunized with 1.5×10 ⁷ PDL1-CHOK1 cells. Immunized mice were re-immunized with 1.5×10 ⁷ PDL1-CHOK1 cells 14 and 33 days after the first immunization, respectively. Sera from immunized mice were tested by ELISA to select mice that produce antibodies that bind to PD-L1 protein. Briefly, 1 μg/ml human PD-L1 protein in PBS was added at 100 μl per well to coat the microtiter plate and left overnight at room temperature (RT) before blocking with 100 μl per well of 5% BSA. did. A dilution of plasma from immunized mice was added to each well and incubated at room temperature for 1-2 hours. Plates were washed with PBS/Tween and then incubated with horseradish peroxidase (HRP) conjugated anti-mouse IgG antibody for 1 hour at room temperature. After washing, plates were developed with ABTS substrate and analyzed by spectrophotometer at OD 405 nm. 54 days after immunization, mice with a sufficient titer of anti-PDL1 IgG were boosted with 50 μg human PDL1-Fc protein. The resulting mice were used for fusions. Hybridoma supernatants were tested for anti-PD-L1 IgG by ELISA.

Hybridoma clones HL1210-3, HL1207-3, HL1207-9 and HL1120-3 were selected for further analysis. The amino acid and polynucleotide sequences of the HL1210-3 variable region are provided in Table 5 below.
[Table 5 HL1210-3 variable region sequence]

[Example 2: Binding activity of HL1210-3 mouse monoclonal antibody to human PD-L1]
To assess the binding activity of hybridoma clone HL1210-3, an ELISA test was performed on the mAb purified from this clone. Briefly, 100 μl per well of 0.1 μg/ml human PD-L1-Fc protein in PBS was added to coat the microtiter plate, left overnight at 4° C., and then 100 μl per well of 5% BSA was added. Blocked with. A 3-fold dilution of HL1210-3 antibody starting from 0.2 μg/ml was added to each well and incubated at room temperature for 1-2 hours. Plates were washed with PBS/Tween and then incubated with goat anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-630 nm. As shown in FIG. 1, HL1210-3 can bind human PD-L1 with high activity (EC ₅₀ =5.539 ng/ml).

[Example 3: Block of human PD-L1 binding to receptor PD-1 by HL1210-3 mouse mAb]
[Receptor blocking assay using recombinant human PD-L1]
To evaluate the blocking effect of HL1210-3 mouse mAb on the binding of recombinant human PD-L1 to receptor PD-1, an ELISA-based receptor blocking assay was utilized. Briefly, 100 μl per well of 1 μg/ml human PD-L1-Fc protein in PBS was added to coat the microtiter plate, left overnight at 4° C., and then 100 μl per well of 5% BSA was used. Blocked. 50 μl of biotin-labeled human PD-1-Fc protein and a 3-fold dilution of HL1210-3 antibody starting at 2 μg/ml in 50 μl were added to each well and incubated at 37° C. for 1 hour. Plates were washed with PBS/Tween and then incubated with streptavidin-HRP for 1 hour at 37°C. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-630 nm. As shown in FIG. 2, HL1210-3 can efficiently inhibit the binding of human PD-L1 to human PD1 with IC ₅₀ =0.7835 nM.

[Receptor blocking assay by using mammalian cell-expressed human PD-L1]
A FACS-based receptor blocking assay was used to assess the blocking effect of HL1210-3 mouse mAb on binding of human PD-L1 expressed on mammalian cells to receptor PD-1. Briefly, PDL1-CHOK1 cells were first incubated with 3-fold serially diluted HL1210-3 mouse mAb starting from 20 μg/ml for 1 hour at room temperature. After washing with FACS buffer (PBS+2% FBS), biotin-labeled huPD-1 was added to each well and incubated at room temperature for 1 hour. Next, streptavidin-PE was added to each well, and post-washing was performed twice with a FACS buffer for 0.5 hours. Mean fluorescence intensity (MFI) of PE was evaluated by FACSAriaIII. As shown in FIG. 3, the HL1210-3 antibody can inhibit the binding of PD-1 to PD-L1 expressed on mammalian cells with high efficiency (IC50: 2.56 nM, maximum inhibition rate: 92.6%). ).
Inhibition rate (%)=(1-MFI of test antibody/MFI of control carrier)×100%

[Example 4: Human T cell immune response promoted by HL1210-3 mouse mAb]
To assess the effects of HL1210-3 mouse mAb, the response of human T cells in a mixed lymphocyte reaction environment was examined. Human DC were differentiated from CD14+ monocytes for 7 days in the presence of GM-CSF and IL-4. CD4+ T cells isolated from another donor were then co-cultured with a dilution series of DC and anti-PD-L1 blocking antibody. Culture supernatants were assayed for IFNγ production 5 days after inoculation. As a result, it was shown that the HL1210-3 antibody can promote the production of IFNγ in a dose-dependent manner. This suggests that anti-PD-L1 antibody can promote human T cell response (FIG. 4).

[Example 5: Binding affinity of HL1210-3 mouse mAb]
The capture method was used to test HL1210-3 antibody binding to recombinant PD-L1 protein (human PD-L1-his taq) using BIACORE™. HL1210-3 mouse mAb were collected using anti-mouse Fc antibody coated on CM5 chip. To the collected antibody, a dilution series of human PD-L1-his taq protein was injected for 3 minutes at a flow rate of 25 μg/ml. The antigen was dissociated for 900 seconds. All experiments were performed on a Biacore T200®. Data analysis was performed using Biacore T200® evaluation software. The results are shown in Figure 5 and Table 6 below.
[Table 6 Binding rate of HL1210-3 to recombinant human PD-L1]

[Example 6: Humanization of HL1210-3 mouse mAb]
The humanized mAb was generated using the mAb HL1210-3 variable region gene. In the first step of this process, the MAb HL1210-3 VH and VK amino acid sequences were compared to available databases of human Ig gene sequences to find the highest overall germline human germline Ig gene sequences. did. For the light chains, the closest human matches are the O18/Jk2 and KV1-39*01/KJ2*04 genes. For the heavy chain, the closest human match is the VH3-21 gene. The VH3-11, VH3-23, VH3-7*01 and VH3-48 genes were also selected due to their high concordance.

Next, CDR1 (SEQ ID NO: 4), 2 (SEQ ID NO: 5) and 3 (SEQ ID NO: 6) of the HL1210-3 light chain are the frames of the O18/Jk2 and KV1-39*01/KJ2*04 genes. CDR1 (SEQ ID NO: 1), 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3) sequences of VH3-21, VH3-11, VH3-23 of HL1210-3 VH transplanted to the work sequence and The humanized variable domain sequences were designed to be grafted onto the framework sequences of the VH3-48 or VH3-7*01 genes. A three-dimensional model was then generated to determine if there were any framework positions that could affect binding and/or CDR morphology by replacing mouse amino acids with human amino acids. For the light chain, 22S, 43S, 60D, 63T and 42Q (Kabat numbering, see Table 7) in the framework were identified. In the case of the heavy chain, 1E, 37V, 40T, 44S, 49A, 77N, 91I, 94R and 108T in the framework were involved in the back mutation.
[Table 7 Humanized Design]

The amino acid and nucleotide sequences of some humanized antibodies are listed in Table 8 below.
[Table 8 Humanized antibody sequences (bold letters indicate CDRs)]

The humanized VH and VK genes were generated synthetically and then cloned into vectors containing human γ1 and human κ constant domains, respectively. The combination of human VH and human VK formed 40 humanized antibodies (see Table 9).
[Table 9 Humanized antibody having VH and VL regions]

[Example 7: Antigen binding property of humanized PD-L1 antibody]
[Binding properties of recombinant human PD-L1]
An ELISA test was performed on the humanized antibody to evaluate the antigen binding activity. Briefly, 100 μl per well of 0.1 μg/ml human PD-L1-Fc protein in PBS was added to coat the microtiter plate, left overnight at 4° C., and then 100 μl per well of 5% BSA was added. Blocked with. A 5-fold dilution of humanized antibody starting at 10 μg/ml was added to each well and incubated at room temperature for 1-2 hours. Plates were washed with PBS/Tween and then incubated with goat anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-630 nm. As shown in Figure 6, all humanized antibodies show comparable binding potency to human PD-L1 contacting chimeric antibodies.

[Binding properties of human PD-L1 expressed in mammalian cells]
To assess antigen binding properties, humanized antibodies were analyzed by FACS for binding to PD-L1 expressed in mammalian cells. Briefly, PDL1-CHOK1 cells were first incubated with a 5-fold serial dilution of humanized antibody starting at 2 μg/ml for 1 hour at room temperature. After washing with FACS buffer (PBS+2% FBS), Alexa488-anti-human IgG antibody was added to each well and incubated at room temperature for 1 hour. The MFI of Alexa488 was evaluated by FACSAriaIII. As shown in FIG. 7, all humanized antibodies can bind PD-L1 expressed in mammalian cells with the same high efficiency as the chimeric antibody.

To investigate the binding rate of humanized antibodies, affinity ranking was performed in this example by using Octet Red 96. As shown in Table 10, Hu1210-3, Hu1210-8, Hu1210-9, hu1210-14, Hu1210-17, hu1210-1 and Hu1210-22 show higher affinity comparable to the chimeric antibody.
[Table 10 Affinity Ranking of Humanized Antibodies]

[Overall Kinetic Affinity of Humanized Antibodies by Biacore®]
The capture method was used to test humanized antibody binding to recombinant PD-L1 protein (human PD-L1-his taq) using BIACORE™. HL1210-3 mouse mAb were collected using anti-mouse Fc antibody coated on CM5 chip. To the collected antibody, a dilution series of human PD-L1-his taq protein was injected for 3 minutes at a flow rate of 25 μg/ml. The antigen was dissociated for 900 seconds. All experiments were performed on a Biacore T200®. Data analysis was performed using Biacore T200® evaluation software and is shown in Table 11 below.
[Table 11 Affinity by Biacore]

[Inter-species activity]
To assess the binding of humanized antibodies to huPD-L1, mouse PD-L1, rat PD-L1, rhesus PD-L1, an ELISA test was performed on the antibodies. Briefly, 1 μg/ml of human, mouse, rat and rhesus PD-L1-Fc protein in PBS was added at 100 μl per well to coat the microtiter plate and left overnight at 4° C., then 100 μl/well Blocked with 5% BSA. A 3-fold dilution of humanized antibody starting at 1 μg/ml was added to each well and incubated at room temperature for 1-2 hours. Plates were washed with PBS/Tween and then incubated with goat anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-630 nm. The Hu1210-41 antibody can bind rhesus PD-L1 with low affinity and not rat and mouse PD-L1 (FIG. 8).

[Family member specificity]
To assess the binding of humanized anti-PD-L1 antibody to human B7 family and other immune checkpoints, B7-H1 (PD-L1), B7-DC, B7-1, B7-2, by ELISA, Antibodies were evaluated for binding to B7-H2, PD-1, CD28, CTLA4, ICOS and BTLA. As shown in FIG. 9, the Hu1210-41 antibody can specifically bind only to B7-H1 (PD-L1).

[Example 8: Activity of human PD-L1 on PD-1 blocked by humanized antibody]
[Cell-based receptor blocking assay]
A FACS-based receptor blocking assay was utilized to assess the blocking effect of humanized antibodies on the binding of human PD-L1 expressed on mammalian cells to the receptor PD-1. Briefly, PDL1-CHOK1 cells were first incubated with 3-fold serially diluted HL1210-3 mouse mAb starting from 20 μg/ml for 1 hour at room temperature. After washing with FACS buffer (PBS+2% FBS), biotin-labeled huPD-1 was added to each well and incubated at room temperature for 1 hour. Next, streptavidin-PE was added to each well and post-washing was carried out twice with FACS buffer for 0.5 hours. Mean fluorescence intensity (MFI) of PE was evaluated by FACSAriaIII.
Inhibition rate (%)=(1-MFI of test antibody/MFI of control carrier)×100%

As shown in Table 12 below, the Hu1210-3, Hu1210-9, Hu1210-8, Hu1210-14, Hu1210-17, Hu1210-19 and Hu1210-22 antibodies were found to bind PD-L1 to PD-1. The block is as effective as the chimeric antibody.
[Table 12 PD-1 receptor blocking assay]

[Receptor blocking assay using recombinant human PD-L1]
For human PD-L1, there are two receptors, PD-1 and B7-1. To investigate the blocking properties of humanized PD-L1 antibodies against these two proteins, a protein-based receptor blocking assay was utilized here. Briefly, 100 μl per well of 1 μg/ml human PD-L1-Fc protein in PBS was added to coat the microtiter plate, left overnight at 4° C., and then 200 μl per well of 5% BSA was used. And blocked at 37° C. for 2 hours. 50 μl of biotin-labeled human PD-1-Fc or B7-1v protein and a 5-fold dilution of PD-L1 antibody starting from 50 μl at 100 nM was added to each well and incubated at 37° C. for 1 hour. Plates were washed with PBS/Tween and then incubated with streptavidin-HRP for 1 hour at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. As shown in FIGS. 10 and 11, Hu1210-41 can efficiently inhibit human PD-L1n binding to human PD1 and B7-1.

[Example 9: Human T cell immune reaction promoted by humanized antibody]
[Mixed lymphocyte reaction assay]
The response of human T cells in a mixed lymphocyte reaction environment was examined to evaluate the in vitro function of humanized antibodies. Human DC were differentiated from CD14+ monocytes for 7 days in the presence of GM-CSF and IL-4. CD4+ T cells isolated from another donor were then co-cultured with a dilution series of DC and anti-PD-L1 blocking antibody. Culture supernatants were assayed for IL-2 and IFNγ production 5 days after inoculation. The results show that the Hu1210-8, Hu1210-9, Hu1210-16 and Hu1210-17 antibodies can enhance the production of IL-2 and IFNγ in a dose-dependent manner, and anti-PD-L1 antibody induces a human T cell response. Suggest that it can be promoted.

[CMV recall assay]
To assess the in vitro function of humanized antibodies, human T cell responses were examined in a CMV recall assay. Human PBMCs were stimulated with 1 μg/ml CMV antigen in the presence of serially diluted humanized antibody. As shown in FIGS. 12 and 13, Hu1210-40, Hu1210-41 and Hu1210-17 are capable of promoting IFNγ production in a dose-dependent manner.

[Example 10: Inhibition of tumor growth by anti-PD-L1 mAb]
Cells derived from the human lung adenocarcinoma cell line HCC827 are transplanted into NOD scid gamma (NSG) mice. NSG mice are deficient in NOD scid gamma and are the most immunodeficient mice, and thus are ideal recipients of human tumor cells and PBMC transplants. Ten days after transplantation, human PBMCs are transplanted into tumor-bearing mice. Approximately 20 days after transplantation, when the tumor volume reaches 100 to 150 mm ³ , mice are administered 5 mg/kg of PD-L1 antibody every other day. Tumor volume is monitored every other day in conjunction with antibody administration. As shown in FIG. 14, Hu1210-31 can inhibit tumor growth by 30% at 5 mg/kg. The Hu1210-41 antibody can inhibit tumor growth in a dose-dependent manner, while tumor weight is also suppressed by the Hu1210-41 antibody in a dose-dependent manner (Fig. 15).

[Example 11 Computer simulation of further modification and optimization of humanized antibody]
It was envisioned that by altering specific amino acid residues in the CDR or framework regions, the activity and/or stability of the antibody may be further improved or retained. Computational tools (Vector NTI, available at www.ebi.ac.uk/tools/msa/clustalo/) were used to test variants for structural, conformational, and functional properties. The potential ones (inside the CDR regions) are listed in the table below.
Table 13 VH and VL CDRs and variants suitable for incorporation into humanized antibodies
Underline: Hotspot mutant residue and its substitution

[Example 12: Identification of PD-L1 epitope]
This study was performed to identify the amino acid residues involved in the binding of PD-L1 to the antibodies of this disclosure.

An alanine scanning library of PD-L1 was constructed. Briefly, 217 mutant clones of PD-L1 were generated on the protein molecular platform of Integral Molecular. Hu1210-41 Fab binding to each variant in the PD-L1 mutation library was determined in duplicate by high throughput flow cytometry. At each raw data point, background fluorescence was subtracted and normalized to PD-L1 wild type (WT) reactivity. Mean binding values were plotted as a function of expression for each PD-L1 variant (control: anti-PD-L1 mAb reactivity). To identify tentatively important clones (circles with crosses), it is said that 70% or more of the WT binds to the control mAb and less than 30% of the WT having reactivity with Hu1210-41 Fab. A threshold (dashed line) was applied (Fig. 16). PDL1 Y134, K162 and N183 were identified as the residues required for Hu1210-41 binding. The low reactivity of the N183A clone to the Hu1210-41 Fab suggests that it contributes energetically to Hu1210-41 binding, with small contributions of Y134 and K162.

As shown in FIG. 17, important residues (spheres) were identified on the three-dimensional PD-L1 structure (PDB ID# 5JDR, Zhang et al., 2017). Therefore, these residues, Y134, K162 and N183, constitute the epitope of PD-L1 responsible for binding to the antibodies of various embodiments of the present disclosure.

Interestingly, note that Y134, K162 and N183 are all located in the IgC domain of the PD-L1 protein. The extracellular portion of both PD-1 and PD-L1 has IgV and IgC domains. PD-L1 is generally known to bind to PD-1 through binding between IgV domains. However, unlike such conventional antibodies, Hu1210-41 binds to the IgC domain. It was expected to be ineffective in inhibiting PD-1/PD-L1 binding. Surprisingly, this different epitope of Hu1210-41 likely contributes to the excellent activity of Hu1210-41.

[Example 13. Antibody design of anti-PDL1 antibody]
Examples 13-17 attempted to use mutagenesis to identify further improved antibodies based on Hu1210-41.

A fusion protein of activation-induced cytidine deaminase (AID) and nuclease-inactive clustered regularly interspersed short palindromic repeats (CRISPR)-related protein 9 (dCas9) was used for high-throughput screening of functional variants in 293 T cells. .. Briefly, a single guide (sg) RNA that recognizes the antibody's 6 CDRs can direct the dCas9-AID fusion protein to the antibody's 6 CDRs to induce mutations in the CDR regions. The mutant antibody was displayed on the cell surface of 293 cells. The resulting cells showed better avidity than the non-mutated control and were FACS sorted for subsequence sequencing. The S60 to R mutation in CDRH2 was identified as likely to have a good effect on the antibody.

To assess the antigen binding properties of S60R(CDRH2) variants, antibodies were analyzed by FACS for binding to PD-L1 expressed in mammalian cells. Briefly, PD-L1 Raji cells were first incubated for 1 hour at room temperature with 5-fold serial dilutions of humanized antibody starting at 2 μg/ml. After washing with FACS buffer (PBS+2% FBS), Alexa488-anti-human IgG antibody was added to each well and incubated at room temperature for 1 hour. The MFI of Alexa488 was evaluated by FACSAriaIII. As shown in FIG. 18, the S60R mutant binds PD-L1 expressed in mammalian cells with higher efficiency, which is more potent than the parent antibody Hu1210-41. The S60R mutant was then used as the parental antibody for further mutation analysis below and was designated "WT".

Four sub-libraries were constructed for antibody design of anti-PD-L1 monoclonal antibodies using either of the following strategies. In strategy 1, mutagenesis of the heavy chain variable domain VH CDR3 or VL-CDR3 was performed by highly random mutations. In strategy 2, consisting of (VH-CDR3, VL-CDR3, and VL-CDR1) or (VH-CDR1, VH-CDR2, and VL-CDR2) by controlling the mutation rate and performing CDR walking Two CDR combinatorial libraries were generated.

Biopanning: The phage panning method was adapted by shortening the incubation/binding time before severe washing conditions. Briefly, 100 μl of magnetic streptavidin beads (Invitrogen®, USA) was blocked with 1 ml of MPBS for 1 hour at room temperature. In a separate tube, library phage were pre-incubated in 1 ml MPBS with 100 μl of magnetic streptavidin beads (5×10̂11-12 each time) to remove unwanted binder. Phage and beads were separated using a magnetic particle concentrator. Biotinylated PD-L1 protein was added to the phage, incubated at room temperature for 2 hours and mixed gently using an overhead shaker. The beads with phage from the solution were separated and the supernatant discarded in a magnetic particle concentrator. The beads were washed with fresh wash buffer 10 times with PBST and 10 times with PBS (pH 7.4). 0.8 ml of 0.25% trypsin in PBS (Sigma, USA) was added and incubated at 37°C for 20 minutes to elute the phage. The resulting phage was titrated and collected for the next round of panning, with decreasing antigen concentration with each additional round.

[ELISA screening and on/off rate ranking]
Clones were selected and derived from the desired panning output. A phage ELISA was performed for the primary screen. Positive clones were analyzed by sequencing. Unique hotspots have been discovered. Table 14 shows the identified mutations. As shown below, the FGK residue in CDRH3 is a hotspot residue that produces improved antibodies.
[Table 14. Mutations in CDR]
*WT differs from Hu1210-41 by S60R (Kabat numbering) substitution in the heavy chain to improve affinity.

The amino acid sequences of the variable regions of these antibodies are shown in Table 15 below.
[Table 15. Antibody sequence]

[Example 14. Antigen-binding properties of PD-L1 antibody]
As shown in Tables 14 and 15, a total of 9 unique clones were revealed and converted to full length IgG.

[Binding properties of recombinant human PD-L1]
An ELISA test was performed on the antibodies to assess antigen binding activity. Briefly, 100 μl per well of 2 μg/ml human PD-L1-Fc protein in PBS was added to coat the microtiter plate, left overnight at 4° C., and then 100 μl per well of 5% BSA was used. Blocked. A 4-fold dilution of humanized antibody starting at 10 μg/ml was added to each well and incubated at room temperature for 1-2 hours. Plates were washed with PBS/Tween and then incubated with goat anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450-630 nm. As shown in Figure 19, all humanized antibodies show superior binding potency to human PD-L1, with B6 and C3 behaving better than the parental clone WT.

[Binding properties of human PD-L1 expressed in mammalian cells]
Antibodies were analyzed by FACS for binding to PD-L1 expressed in mammalian cells to assess antigen binding properties. Briefly, PDL1-Raji cells were first incubated at room temperature for 1 hour with 5-fold serial dilutions of humanized antibody starting at 2 μg/ml. After washing with FACS buffer (PBS+2% FBS), Alexa488-anti-human IgG antibody was added to each well and incubated at room temperature for 1 hour. The MFI of Alexa488 was evaluated by FACSAriaIII. As shown in FIG. 20, B6 bound PD-L1 expressed on mammalian cells with high efficiency, which was more potent than the parental antibody WT.

[Affinity Ranking of Humanized Antibodies by Biacore]
To investigate the binding kinetics of humanized antibodies, Biacore® was used in this example to perform affinity ranking. As shown in Table 16, B6, C3, C6, A1 and A3 showed better affinity than the parent antibody WT.
[Table 16. Affinity ranking]
Example 15. Anti-PDL1 antibody cell-based function

To test the ability of anti-PDL1 antibodies to stimulate T cell responses, Jurkat cells expressing hPD-1 were used. Briefly, Jurkat is a human T cell leukemia cell line capable of producing IL2 in response to TCR stimulation. In this assay, Jurkat cells transfected with the human PD-1 gene by lentivirus were used as responder cells. Raji-PDL1 cells were used as antigen presenting cells (APC). Staphylococcal enterotoxin (SE) is used to stimulate TCR signals. In this system, ectopically expressed huPDL1 can suppress SE-stimulated IL-2 production by Jurkat cells, while anti-PDL1 antibody can reverse IL-2 production. That is, APC (2.5×10 ⁴ ) was co-cultured with PD-1 expressing Jurkat T cells (1×10 ⁵ ) in the presence of SE stimulation. At the beginning of the culture, anti-PDL1 antibody (starting from 100 nM and serially diluted 1:4 at 8 points) was added. After 48 hours, culture supernatants were evaluated for IL2 production by ELISA. As shown in Figure 21, the B6 monoclonal antibody was more potent than the parental antibody WT.

[Example 16. Mixed lymphocyte reaction]
The response of human T cells in a mixed lymphocyte reaction environment was examined to evaluate the in vitro function of PDL1 antibodies. Briefly, human DC were differentiated from CD14+ monocytes for 7 days in the presence of GM-CSF and IL-4. CD4+ T cells isolated from another donor were then co-cultured with a dilution series of DC and anti-PD-L1 blocking antibody. Culture supernatants were assayed for IFNγ production 5 days after inoculation. The results (FIG. 22) showed that the B6 antibody was more potent than the parental antibody WT in promoting IFNγ production.

[Example 17. In vivo efficacy of PDL1 antibody in MC38 syngeneic model]
To assess the effect of PDL1 on tumor growth, the PDL1 humanized MC38 syngeneic tumor model was applied. In this model, the human PDL1 gene was expressed in mouse MC38 cells, while the extracellular domain of the mouse PDL1 gene was replaced with the human PDL1 gene. In this regard, the efficacy of human PDL1 antibody on tumor growth could be evaluated in this PDL1 gene humanized MC38 syngeneic tumor model. HuPDL1 MC38 cells were subcutaneously inoculated into PDL1 humanized mice. When tumors reached a volume of 100-150 ^3, the parent antibody WT and B6 antibody, twice a week 3 mg / kg, administered intraperitoneally, was given 6 times in total. The results (Figure 23) showed that the B6 antibody was more potent than the parental antibody WT at 19-26 days.

This disclosure is not intended to be limited in scope by the particular embodiments described, which are intended to be illustrative of one of the individual aspects of the disclosure, but any functionally equivalent composition. Articles or methods are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed methods and compositions without departing from the spirit or scope of the disclosure. Accordingly, this disclosure is intended to cover modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents.

All publications and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Be incorporated into.

Claims (78)

An antibody or antibody fragment,
The antibody or antibody fragment has specificity for human PD-L1 protein,
(A) SEQ ID NO: 1 or a VH CDR1 comprising the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:1
(B) SEQ ID NO:116 or a VH CDR2 comprising the amino acid sequence of a variant of SEQ ID NO:116 containing 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:116,
(C) a VH CDR3 comprising SEQ ID NO:117 or the amino acid sequence of a variant of SEQ ID NO:117 containing 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:117, A VH CDR3 in which the second amino acid residue is Leu,
(D) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, or a variant of SEQ ID NO:4 having 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:4,
(E) SEQ ID NO:5, or a VL CDR2 comprising the amino acid sequence of a variant of SEQ ID NO:5 having 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:5, and
(F) VL CDR3 comprising SEQ ID NO:6, or the amino acid sequence of a variant of SEQ ID NO:6 having 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:6.
An antibody or antibody fragment comprising: The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:1 has a single amino acid substitution at one of amino acid residues 1, 2 and 5. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:1 is selected from the group consisting of SEQ ID NO:61-67. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:116 has a single amino acid substitution at one of amino acid residues 7 and 8. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:116 is selected from the group consisting of SEQ ID NO:118-127, 2 and 68-77. The antibody or antibody fragment according to claim 1, wherein the third amino acid residue of the VH CDR3 is Pro. The antibody or antibody fragment according to claim 1, wherein the fourth amino acid residue of VH CDR3 is Trp. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO: 117 has a single amino acid substitution at one of amino acid residues 1, 5 and 6. The antibody or antibody fragment of claim 1, wherein said variant of SEQ ID NO:117 is selected from the group consisting of SEQ ID NO:128-139. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:4 has a single amino acid substitution at amino acid residue 3. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:4 is selected from the group consisting of SEQ ID NO:91-92. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:5 has a single amino acid substitution at one of amino acid residues 1-6. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:5 is selected from the group consisting of SEQ ID NO:93-105. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO: 6 has a single amino acid substitution at one of amino acid residues 1 and 2. The antibody or antibody fragment of claim 1, wherein the variant of SEQ ID NO:6 is selected from the group consisting of SEQ ID NO:140 and 106-111. The VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 116, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 117, and the VL CDR1 is The antibody or antibody fragment according to claim 1, which comprises the amino acid sequence of SEQ ID NO: 4, said VL CDR2 comprises the amino acid sequence of SEQ ID NO: 5, and said VL CDR3 comprises the amino acid sequence of SEQ ID NO: 6. .. 17. The antibody or antibody fragment of claim 16, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:149 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:150. An antibody or antibody fragment,
The antibody or antibody fragment has specificity for human PD-L1 protein,
(A) SEQ ID NO: 1 or a VH CDR1 comprising the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:1
(B) SEQ ID NO:116 or a VH CDR2 comprising the amino acid sequence of a variant of SEQ ID NO:116 having 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:116,
(C) SEQ ID NO:3, or a VH CDR3 comprising the amino acid sequence of a variant of SEQ ID NO:3 with 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:3,
(D) a SEQ ID NO: 4 or a VL CDR1 comprising the amino acid sequence of a variant of SEQ ID NO: 4 with 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:4,
(E) SEQ ID NO:5, or a VL CDR2 comprising the amino acid sequence of a variant of SEQ ID NO:5 containing 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:5, and
(F) comprising SEQ ID NO: 140, or the amino acid sequence of a variant of SEQ ID NO: 140 with 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:140, at least (i) VL CDR3, wherein amino acid residue 4 of said VL CDR3 is Ser, (ii) amino acid residue 5 of said VL CDR3 is Asp, or (iii) amino acid residue 6 of said VL CDR3 is Ala.
An antibody or antibody fragment comprising: 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO: 1 has a single amino acid substitution at one of amino acid residues 1, 2 and 5. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:1 is selected from the group consisting of SEQ ID NO:61-67. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:116 has a single amino acid substitution at one of amino acid residues 7 and 8. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:116 is selected from the group consisting of SEQ ID NO:118-127, 2 and 68-77. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:3 has a single amino acid substitution at one of amino acid residues 1, 5 and 6. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:3 is selected from the group consisting of SEQ ID NO:117 and 128-139. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:4 has a single amino acid substitution at amino acid residue 3. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:4 is selected from the group consisting of SEQ ID NO:91-92. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:5 has a single amino acid substitution at one of amino acid residues 1-6. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:5 is selected from the group consisting of SEQ ID NO:93-105. 19. The antibody or antibody fragment of claim 18, wherein amino acid residue 4 of VL CDR3 is Ser. 19. The antibody or antibody fragment of claim 18, wherein amino acid residue 5 of VL CDR3 is Asp. 19. The antibody or antibody fragment of claim 18, wherein amino acid residue 6 of VL CDR3 is Ala. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:140 has a single amino acid substitution at one of amino acid residues 1 and 2. 19. The antibody or antibody fragment of claim 18, wherein the variant of SEQ ID NO:140 is selected from the group consisting of SEQ ID NO:161-166. The VH CDR1 includes the amino acid sequence of SEQ ID NO: 1, the VH CDR2 includes the amino acid sequence of SEQ ID NO: 116, the VH CDR3 includes the amino acid sequence of SEQ ID NO: 3, and the VL CDR1 is The antibody or antibody fragment of claim 1, comprising the amino acid sequence of SEQ ID NO:4, said VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and said VL CDR3 comprising the amino acid sequence of SEQ ID NO:140. .. 35. The antibody or antibody fragment of claim 34, which comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:159 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:160. An antibody or antibody fragment,
The antibody or antibody fragment has specificity for human PD-L1 protein,
(A) SEQ ID NO: 1 or a VH CDR1 comprising the amino acid sequence of the variant of SEQ ID NO: 1 containing 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:1
(B) SEQ ID NO:116 or a VH CDR2 comprising the amino acid sequence of a variant of SEQ ID NO:116 containing 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:116,
(C) SEQ ID NO:3, or a VH CDR3 comprising the amino acid sequence of the variant of SEQ ID NO:3 containing 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:3,
(D) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, or a variant of SEQ ID NO:4 having 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:4,
(E) SEQ ID NO:5, or a VL CDR2 comprising the amino acid sequence of a variant of SEQ ID NO:5 having 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:5, and
(F) VL CDR3 comprising SEQ ID NO:6, or the amino acid sequence of a variant of SEQ ID NO:6 having 1, 2 or 3 substitutions, deletions or insertions as compared to SEQ ID NO:6.
An antibody or antibody fragment comprising: 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO: 1 has a single amino acid substitution at one of amino acid residues 1, 2 and 5. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:1 is selected from the group consisting of SEQ ID NO:61-67. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:116 has a single amino acid substitution at one of amino acid residues 7 and 8. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO: 116 is selected from the group consisting of SEQ ID NO: 118-127, 2 and 68-77. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:3 has a single amino acid substitution at one of amino acid residues 1, 5 and 6. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:3 is selected from the group consisting of SEQ ID NO:117 and 128-139. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:4 has a single amino acid substitution at amino acid residue 3. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:4 is selected from the group consisting of SEQ ID NO:91-92. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:5 has a single amino acid substitution at one of amino acid residues 1-6. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:5 is selected from the group consisting of SEQ ID NO:93-105. 37. The antibody or antibody fragment of claim 36, wherein amino acid residue 4 of VL CDR3 is Ser. 37. The antibody or antibody fragment of claim 36, wherein amino acid residue 5 of VL CDR3 is Asp. 37. The antibody or antibody fragment of claim 36, wherein amino acid residue 6 of VL CDR3 is Ala. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:6 has a single amino acid substitution at one of amino acid residues 1 and 2. 37. The antibody or antibody fragment of claim 36, wherein the variant of SEQ ID NO:6 is selected from the group consisting of SEQ ID NO:140 and 106-111. The VH CDR1 includes the amino acid sequence of SEQ ID NO: 1, the VH CDR2 includes the amino acid sequence of SEQ ID NO: 116, the VH CDR3 includes the amino acid sequence of SEQ ID NO: 3, and the VL CDR1 is 37. The antibody or antibody fragment of claim 36, which comprises the amino acid sequence of SEQ ID NO:4, said VL CDR2 comprises the amino acid sequence of SEQ ID NO:5, and said VL CDR3 comprises the amino acid sequence of SEQ ID NO:6. .. 53. The antibody or antibody fragment of claim 52, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:141 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:142. 54. The antibody or antibody fragment of any one of claims 1-53, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. 54. The antibody or antibody fragment according to any one of claims 1 to 53, wherein the antibody or antibody fragment is a chimeric antibody or a humanized antibody. According to Kabat numbering,
(A) Ser at position 44,
(B) Ala at position 49,
(C) Ala at position 53,
(D) Ile at position 91,
(E) Glu at position 1,
(F) Val at position 37,
(G) Thr at position 40,
(H) Val at position 53,
(I) Glu at position 54,
(J) Asn at position 77,
(K) Arg at position 94, and
(L) Thr at position 108, and
56. The antibody or antibody fragment of claim 55, which comprises a heavy chain variable region comprising one or more amino acid residues selected from the group consisting of: A heavy chain variable comprising (a) Ser at position 44, (b) Ala at position 49, (c) Ala at position 53, and/or (d) Ile at position 91, and combinations thereof, according to Kabat numbering. 56. The antibody or antibody fragment of claim 55, which comprises a region. According to Kabat numbering,
(A) Ser at position 22,
(B) Gln at position 42,
(C) Ser at position 43,
(D) Asp at position 60, and
(E) Thr at position 63, and
56. The antibody or antibody fragment of claim 55, comprising a light chain variable region comprising one or more amino acid residues selected from the group consisting of combinations thereof. An antibody which is a bispecific antibody and comprises the antibody fragment according to any one of claims 1 to 53 and a second antigen-binding fragment having specificity for molecules on immune cells. A bispecific antibody, wherein the molecule is PD-1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL. 60. The antibody of claim 59, selected from the group consisting of: GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM, BTLA, KIR and CD47. A bispecific antibody, wherein the antibody fragment and the second antigen-binding fragment are each independently selected from a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody. Item 59. The antibody according to Item 59. 60. The antibody of claim 59 which is a bispecific antibody further comprising an Fc fragment. 63. A composition comprising the antibody or antibody fragment of any one of claims 1-62 and a pharmaceutically acceptable carrier. 63. A polynucleotide encoding one of the polypeptide chains of the antibody or antibody fragment of any one of claims 1-62. 63. An isolated cell comprising one or more polynucleotides encoding the antibody or antibody fragment of any one of claims 1-62. 63. A method of treating cancer or infection in a patient in need thereof, comprising administering to said patient an effective amount of an antibody or antibody fragment according to any one of claims 1-62. 67. The method of claim 66, wherein the cancer is a solid tumor. The cancer, bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, digestive organ cancer, 67. The method of claim 66, selected from the group consisting of gastric cancer, esophageal cancer, ovarian cancer, renal cancer, malignant melanoma, prostate cancer, and thyroid cancer. 67. The method of claim 66, further comprising administering a second cancer therapeutic agent to the patient. 67. The method of claim 66, wherein the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection. A method of treating a cancer or infection in a patient in need thereof, comprising:
(A) treating cells in vitro with the antibody or antibody fragment according to any one of claims 1 to 44;
(B) administering the treated cells to the patient. 72. The method of claim 71, further comprising the step of isolating the cells from the individual prior to step (a). 72. The method of claim 71, wherein the cells are isolated from the patient. 72. The method of claim 71, wherein the cells are isolated from a donor individual different from the patient. 75. The method of any of claims 71-74, wherein the cells are T cells. 76. The method of claim 75, wherein the T cells are tumor infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof. A method for detecting PD-L1 expression in a sample, comprising:
45. contacting the sample with the antibody or antibody fragment of any one of claims 1 to 44 under conditions in which the antibody or antibody fragment binds to the PD-L1;
Detecting said binding indicative of PD-L1 expression in said sample. 78. The method of claim 77, wherein the sample comprises tumor cells, tumor tissue, infected tissue or blood sample.